Updates

Update 2021.08.11

Most thorough way to readdress differences between original and current analyses is to jsut redo the biomark. To that end, will restart analysis of just CMPm2, excluding other datasets (i.e., LSK, MEP, GMP). Will also keep

Notebook setup

Creating new pipeline using seurat v4.0.2 available 2021.06.08

Load libraries required for Seuratv4

library(dplyr)
library(Seurat)
library(patchwork)
library(ggplot2)
library(clustree)

Set global variables

projectName <- "CMP"
jackstraw.dim <- 40

store session info

sessionInfo.filename <- paste0(projectName, "_sessionInfo.txt")
sink(sessionInfo.filename)
sessionInfo()
sink()
source("../RFunctions/read_10XGenomics_data.R")
source("../RFunctions/PercentVariance.R")
source("../RFunctions/Mouse2Human_idconversion.R")
setwd("../../cellRanger/") # temporarily changing wd only works if you run the entire chunk at once
data_file.list <- read_10XGenomics_data(sample.list = "CMPm2")
object.data <-Read10X(data_file.list)
cmp.object<- CreateSeuratObject(counts = object.data, min.cells = 3, min.genes = 200, project = projectName)

Clean up to free memory

remove(object.data)

Add mitochondrial metadata and plot some basic features

cmp.object[["percent.mt"]] <- PercentageFeatureSet(cmp.object, pattern = "^mt-")
VlnPlot(cmp.object, features = c("nFeature_RNA", "nCount_RNA", "percent.mt"), ncol = 3, pt.size = 0, fill.by = 'orig.ident', )

plot1 <- FeatureScatter(cmp.object, feature1 = "nCount_RNA", feature2 = "percent.mt", group.by = "orig.ident", pt.size = 0.01)
plot2 <- FeatureScatter(cmp.object, feature1 = "nCount_RNA", feature2 = "nFeature_RNA", group.by = "orig.ident", pt.size = 0.01)
plot1 + plot2

Filter data

remove low quality cells require: nFeature_RNA between 200 and 4000 (inclusive) require: percent.mt <=5

print(paste("original object:", nrow(cmp.object@meta.data), "cells", sep = " "))
[1] "original object: 12540 cells"
cmp.object <- subset(cmp.object, 
                                                subset = nFeature_RNA >=200 & 
                                                    nFeature_RNA <= 4000 & 
                                                    percent.mt <= 5
                                                )
print(paste("new object:", nrow(cmp.object@meta.data), "cells", sep = " "))
[1] "new object: 12059 cells"
cmp.object <- NormalizeData(cmp.object, normalization.method = "LogNormalize", scale.factor = 10000)
Performing log-normalization
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|

Find variable features

cmp.object <- FindVariableFeatures(cmp.object, selection.method = "vst", nfeatures = 2000)
Calculating gene variances
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating feature variances of standardized and clipped values
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
top10 <- head(VariableFeatures(cmp.object), 10)
plot1 <- VariableFeaturePlot(cmp.object)
plot2 <- LabelPoints(plot = plot1, points = top10, repel = TRUE)
When using repel, set xnudge and ynudge to 0 for optimal results
plot1 + plot2

Scale data (linear transformation)

We don’t have to worry about comparing library depths, so we’ll just do normalization/Scale data

Regressing out nFeature_RNA, nCount_RNA

  |                                                                                                                                                                                                                      
  |                                                                                                                                                                                                                |   0%
  |                                                                                                                                                                                                                      
  |=                                                                                                                                                                                                               |   0%
  |                                                                                                                                                                                                                      
  |=                                                                                                                                                                                                               |   1%
  |                                                                                                                                                                                                                      
  |==                                                                                                                                                                                                              |   1%
  |                                                                                                                                                                                                                      
  |===                                                                                                                                                                                                             |   1%
  |                                                                                                                                                                                                                      
  |===                                                                                                                                                                                                             |   2%
  |                                                                                                                                                                                                                      
  |====                                                                                                                                                                                                            |   2%
  |                                                                                                                                                                                                                      
  |=====                                                                                                                                                                                                           |   2%
  |                                                                                                                                                                                                                      
  |=====                                                                                                                                                                                                           |   3%
  |                                                                                                                                                                                                                      
  |======                                                                                                                                                                                                          |   3%
  |                                                                                                                                                                                                                      
  |=======                                                                                                                                                                                                         |   3%
  |                                                                                                                                                                                                                      
  |=======                                                                                                                                                                                                         |   4%
  |                                                                                                                                                                                                                      
  |========                                                                                                                                                                                                        |   4%
  |                                                                                                                                                                                                                      
  |=========                                                                                                                                                                                                       |   4%
  |                                                                                                                                                                                                                      
  |=========                                                                                                                                                                                                       |   5%
  |                                                                                                                                                                                                                      
  |==========                                                                                                                                                                                                      |   5%
  |                                                                                                                                                                                                                      
  |===========                                                                                                                                                                                                     |   5%
  |                                                                                                                                                                                                                      
  |===========                                                                                                                                                                                                     |   6%
  |                                                                                                                                                                                                                      
  |============                                                                                                                                                                                                    |   6%
  |                                                                                                                                                                                                                      
  |=============                                                                                                                                                                                                   |   6%
  |                                                                                                                                                                                                                      
  |==============                                                                                                                                                                                                  |   6%
  |                                                                                                                                                                                                                      
  |==============                                                                                                                                                                                                  |   7%
  |                                                                                                                                                                                                                      
  |===============                                                                                                                                                                                                 |   7%
  |                                                                                                                                                                                                                      
  |================                                                                                                                                                                                                |   7%
  |                                                                                                                                                                                                                      
  |================                                                                                                                                                                                                |   8%
  |                                                                                                                                                                                                                      
  |=================                                                                                                                                                                                               |   8%
  |                                                                                                                                                                                                                      
  |==================                                                                                                                                                                                              |   8%
  |                                                                                                                                                                                                                      
  |==================                                                                                                                                                                                              |   9%
  |                                                                                                                                                                                                                      
  |===================                                                                                                                                                                                             |   9%
  |                                                                                                                                                                                                                      
  |====================                                                                                                                                                                                            |   9%
  |                                                                                                                                                                                                                      
  |====================                                                                                                                                                                                            |  10%
  |                                                                                                                                                                                                                      
  |=====================                                                                                                                                                                                           |  10%
  |                                                                                                                                                                                                                      
  |======================                                                                                                                                                                                          |  10%
  |                                                                                                                                                                                                                      
  |======================                                                                                                                                                                                          |  11%
  |                                                                                                                                                                                                                      
  |=======================                                                                                                                                                                                         |  11%
  |                                                                                                                                                                                                                      
  |========================                                                                                                                                                                                        |  11%
  |                                                                                                                                                                                                                      
  |========================                                                                                                                                                                                        |  12%
  |                                                                                                                                                                                                                      
  |=========================                                                                                                                                                                                       |  12%
  |                                                                                                                                                                                                                      
  |==========================                                                                                                                                                                                      |  12%
  |                                                                                                                                                                                                                      
  |==========================                                                                                                                                                                                      |  13%
  |                                                                                                                                                                                                                      
  |===========================                                                                                                                                                                                     |  13%
  |                                                                                                                                                                                                                      
  |============================                                                                                                                                                                                    |  13%
  |                                                                                                                                                                                                                      
  |============================                                                                                                                                                                                    |  14%
  |                                                                                                                                                                                                                      
  |=============================                                                                                                                                                                                   |  14%
  |                                                                                                                                                                                                                      
  |==============================                                                                                                                                                                                  |  14%
  |                                                                                                                                                                                                                      
  |==============================                                                                                                                                                                                  |  15%
  |                                                                                                                                                                                                                      
  |===============================                                                                                                                                                                                 |  15%
  |                                                                                                                                                                                                                      
  |================================                                                                                                                                                                                |  15%
  |                                                                                                                                                                                                                      
  |================================                                                                                                                                                                                |  16%
  |                                                                                                                                                                                                                      
  |=================================                                                                                                                                                                               |  16%
  |                                                                                                                                                                                                                      
  |==================================                                                                                                                                                                              |  16%
  |                                                                                                                                                                                                                      
  |==================================                                                                                                                                                                              |  17%
  |                                                                                                                                                                                                                      
  |===================================                                                                                                                                                                             |  17%
  |                                                                                                                                                                                                                      
  |====================================                                                                                                                                                                            |  17%
  |                                                                                                                                                                                                                      
  |====================================                                                                                                                                                                            |  18%
  |                                                                                                                                                                                                                      
  |=====================================                                                                                                                                                                           |  18%
  |                                                                                                                                                                                                                      
  |======================================                                                                                                                                                                          |  18%
  |                                                                                                                                                                                                                      
  |======================================                                                                                                                                                                          |  19%
  |                                                                                                                                                                                                                      
  |=======================================                                                                                                                                                                         |  19%
  |                                                                                                                                                                                                                      
  |========================================                                                                                                                                                                        |  19%
  |                                                                                                                                                                                                                      
  |=========================================                                                                                                                                                                       |  19%
  |                                                                                                                                                                                                                      
  |=========================================                                                                                                                                                                       |  20%
  |                                                                                                                                                                                                                      
  |==========================================                                                                                                                                                                      |  20%
  |                                                                                                                                                                                                                      
  |===========================================                                                                                                                                                                     |  20%
  |                                                                                                                                                                                                                      
  |===========================================                                                                                                                                                                     |  21%
  |                                                                                                                                                                                                                      
  |============================================                                                                                                                                                                    |  21%
  |                                                                                                                                                                                                                      
  |=============================================                                                                                                                                                                   |  21%
  |                                                                                                                                                                                                                      
  |=============================================                                                                                                                                                                   |  22%
  |                                                                                                                                                                                                                      
  |==============================================                                                                                                                                                                  |  22%
  |                                                                                                                                                                                                                      
  |===============================================                                                                                                                                                                 |  22%
  |                                                                                                                                                                                                                      
  |===============================================                                                                                                                                                                 |  23%
  |                                                                                                                                                                                                                      
  |================================================                                                                                                                                                                |  23%
  |                                                                                                                                                                                                                      
  |=================================================                                                                                                                                                               |  23%
  |                                                                                                                                                                                                                      
  |=================================================                                                                                                                                                               |  24%
  |                                                                                                                                                                                                                      
  |==================================================                                                                                                                                                              |  24%
  |                                                                                                                                                                                                                      
  |===================================================                                                                                                                                                             |  24%
  |                                                                                                                                                                                                                      
  |===================================================                                                                                                                                                             |  25%
  |                                                                                                                                                                                                                      
  |====================================================                                                                                                                                                            |  25%
  |                                                                                                                                                                                                                      
  |=====================================================                                                                                                                                                           |  25%
  |                                                                                                                                                                                                                      
  |=====================================================                                                                                                                                                           |  26%
  |                                                                                                                                                                                                                      
  |======================================================                                                                                                                                                          |  26%
  |                                                                                                                                                                                                                      
  |=======================================================                                                                                                                                                         |  26%
  |                                                                                                                                                                                                                      
  |=======================================================                                                                                                                                                         |  27%
  |                                                                                                                                                                                                                      
  |========================================================                                                                                                                                                        |  27%
  |                                                                                                                                                                                                                      
  |=========================================================                                                                                                                                                       |  27%
  |                                                                                                                                                                                                                      
  |=========================================================                                                                                                                                                       |  28%
  |                                                                                                                                                                                                                      
  |==========================================================                                                                                                                                                      |  28%
  |                                                                                                                                                                                                                      
  |===========================================================                                                                                                                                                     |  28%
  |                                                                                                                                                                                                                      
  |===========================================================                                                                                                                                                     |  29%
  |                                                                                                                                                                                                                      
  |============================================================                                                                                                                                                    |  29%
  |                                                                                                                                                                                                                      
  |=============================================================                                                                                                                                                   |  29%
  |                                                                                                                                                                                                                      
  |=============================================================                                                                                                                                                   |  30%
  |                                                                                                                                                                                                                      
  |==============================================================                                                                                                                                                  |  30%
  |                                                                                                                                                                                                                      
  |===============================================================                                                                                                                                                 |  30%
  |                                                                                                                                                                                                                      
  |===============================================================                                                                                                                                                 |  31%
  |                                                                                                                                                                                                                      
  |================================================================                                                                                                                                                |  31%
  |                                                                                                                                                                                                                      
  |=================================================================                                                                                                                                               |  31%
  |                                                                                                                                                                                                                      
  |==================================================================                                                                                                                                              |  31%
  |                                                                                                                                                                                                                      
  |==================================================================                                                                                                                                              |  32%
  |                                                                                                                                                                                                                      
  |===================================================================                                                                                                                                             |  32%
  |                                                                                                                                                                                                                      
  |====================================================================                                                                                                                                            |  32%
  |                                                                                                                                                                                                                      
  |====================================================================                                                                                                                                            |  33%
  |                                                                                                                                                                                                                      
  |=====================================================================                                                                                                                                           |  33%
  |                                                                                                                                                                                                                      
  |======================================================================                                                                                                                                          |  33%
  |                                                                                                                                                                                                                      
  |======================================================================                                                                                                                                          |  34%
  |                                                                                                                                                                                                                      
  |=======================================================================                                                                                                                                         |  34%
  |                                                                                                                                                                                                                      
  |========================================================================                                                                                                                                        |  34%
  |                                                                                                                                                                                                                      
  |========================================================================                                                                                                                                        |  35%
  |                                                                                                                                                                                                                      
  |=========================================================================                                                                                                                                       |  35%
  |                                                                                                                                                                                                                      
  |==========================================================================                                                                                                                                      |  35%
  |                                                                                                                                                                                                                      
  |==========================================================================                                                                                                                                      |  36%
  |                                                                                                                                                                                                                      
  |===========================================================================                                                                                                                                     |  36%
  |                                                                                                                                                                                                                      
  |============================================================================                                                                                                                                    |  36%
  |                                                                                                                                                                                                                      
  |============================================================================                                                                                                                                    |  37%
  |                                                                                                                                                                                                                      
  |=============================================================================                                                                                                                                   |  37%
  |                                                                                                                                                                                                                      
  |==============================================================================                                                                                                                                  |  37%
  |                                                                                                                                                                                                                      
  |==============================================================================                                                                                                                                  |  38%
  |                                                                                                                                                                                                                      
  |===============================================================================                                                                                                                                 |  38%
  |                                                                                                                                                                                                                      
  |================================================================================                                                                                                                                |  38%
  |                                                                                                                                                                                                                      
  |================================================================================                                                                                                                                |  39%
  |                                                                                                                                                                                                                      
  |=================================================================================                                                                                                                               |  39%
  |                                                                                                                                                                                                                      
  |==================================================================================                                                                                                                              |  39%
  |                                                                                                                                                                                                                      
  |==================================================================================                                                                                                                              |  40%
  |                                                                                                                                                                                                                      
  |===================================================================================                                                                                                                             |  40%
  |                                                                                                                                                                                                                      
  |====================================================================================                                                                                                                            |  40%
  |                                                                                                                                                                                                                      
  |====================================================================================                                                                                                                            |  41%
  |                                                                                                                                                                                                                      
  |=====================================================================================                                                                                                                           |  41%
  |                                                                                                                                                                                                                      
  |======================================================================================                                                                                                                          |  41%
  |                                                                                                                                                                                                                      
  |======================================================================================                                                                                                                          |  42%
  |                                                                                                                                                                                                                      
  |=======================================================================================                                                                                                                         |  42%
  |                                                                                                                                                                                                                      
  |========================================================================================                                                                                                                        |  42%
  |                                                                                                                                                                                                                      
  |========================================================================================                                                                                                                        |  43%
  |                                                                                                                                                                                                                      
  |=========================================================================================                                                                                                                       |  43%
  |                                                                                                                                                                                                                      
  |==========================================================================================                                                                                                                      |  43%
  |                                                                                                                                                                                                                      
  |==========================================================================================                                                                                                                      |  44%
  |                                                                                                                                                                                                                      
  |===========================================================================================                                                                                                                     |  44%
  |                                                                                                                                                                                                                      
  |============================================================================================                                                                                                                    |  44%
  |                                                                                                                                                                                                                      
  |=============================================================================================                                                                                                                   |  44%
  |                                                                                                                                                                                                                      
  |=============================================================================================                                                                                                                   |  45%
  |                                                                                                                                                                                                                      
  |==============================================================================================                                                                                                                  |  45%
  |                                                                                                                                                                                                                      
  |===============================================================================================                                                                                                                 |  45%
  |                                                                                                                                                                                                                      
  |===============================================================================================                                                                                                                 |  46%
  |                                                                                                                                                                                                                      
  |================================================================================================                                                                                                                |  46%
  |                                                                                                                                                                                                                      
  |=================================================================================================                                                                                                               |  46%
  |                                                                                                                                                                                                                      
  |=================================================================================================                                                                                                               |  47%
  |                                                                                                                                                                                                                      
  |==================================================================================================                                                                                                              |  47%
  |                                                                                                                                                                                                                      
  |===================================================================================================                                                                                                             |  47%
  |                                                                                                                                                                                                                      
  |===================================================================================================                                                                                                             |  48%
  |                                                                                                                                                                                                                      
  |====================================================================================================                                                                                                            |  48%
  |                                                                                                                                                                                                                      
  |=====================================================================================================                                                                                                           |  48%
  |                                                                                                                                                                                                                      
  |=====================================================================================================                                                                                                           |  49%
  |                                                                                                                                                                                                                      
  |======================================================================================================                                                                                                          |  49%
  |                                                                                                                                                                                                                      
  |=======================================================================================================                                                                                                         |  49%
  |                                                                                                                                                                                                                      
  |=======================================================================================================                                                                                                         |  50%
  |                                                                                                                                                                                                                      
  |========================================================================================================                                                                                                        |  50%
  |                                                                                                                                                                                                                      
  |=========================================================================================================                                                                                                       |  50%
  |                                                                                                                                                                                                                      
  |=========================================================================================================                                                                                                       |  51%
  |                                                                                                                                                                                                                      
  |==========================================================================================================                                                                                                      |  51%
  |                                                                                                                                                                                                                      
  |===========================================================================================================                                                                                                     |  51%
  |                                                                                                                                                                                                                      
  |===========================================================================================================                                                                                                     |  52%
  |                                                                                                                                                                                                                      
  |============================================================================================================                                                                                                    |  52%
  |                                                                                                                                                                                                                      
  |=============================================================================================================                                                                                                   |  52%
  |                                                                                                                                                                                                                      
  |=============================================================================================================                                                                                                   |  53%
  |                                                                                                                                                                                                                      
  |==============================================================================================================                                                                                                  |  53%
  |                                                                                                                                                                                                                      
  |===============================================================================================================                                                                                                 |  53%
  |                                                                                                                                                                                                                      
  |===============================================================================================================                                                                                                 |  54%
  |                                                                                                                                                                                                                      
  |================================================================================================================                                                                                                |  54%
  |                                                                                                                                                                                                                      
  |=================================================================================================================                                                                                               |  54%
  |                                                                                                                                                                                                                      
  |=================================================================================================================                                                                                               |  55%
  |                                                                                                                                                                                                                      
  |==================================================================================================================                                                                                              |  55%
  |                                                                                                                                                                                                                      
  |===================================================================================================================                                                                                             |  55%
  |                                                                                                                                                                                                                      
  |===================================================================================================================                                                                                             |  56%
  |                                                                                                                                                                                                                      
  |====================================================================================================================                                                                                            |  56%
  |                                                                                                                                                                                                                      
  |=====================================================================================================================                                                                                           |  56%
  |                                                                                                                                                                                                                      
  |======================================================================================================================                                                                                          |  56%
  |                                                                                                                                                                                                                      
  |======================================================================================================================                                                                                          |  57%
  |                                                                                                                                                                                                                      
  |=======================================================================================================================                                                                                         |  57%
  |                                                                                                                                                                                                                      
  |========================================================================================================================                                                                                        |  57%
  |                                                                                                                                                                                                                      
  |========================================================================================================================                                                                                        |  58%
  |                                                                                                                                                                                                                      
  |=========================================================================================================================                                                                                       |  58%
  |                                                                                                                                                                                                                      
  |==========================================================================================================================                                                                                      |  58%
  |                                                                                                                                                                                                                      
  |==========================================================================================================================                                                                                      |  59%
  |                                                                                                                                                                                                                      
  |===========================================================================================================================                                                                                     |  59%
  |                                                                                                                                                                                                                      
  |============================================================================================================================                                                                                    |  59%
  |                                                                                                                                                                                                                      
  |============================================================================================================================                                                                                    |  60%
  |                                                                                                                                                                                                                      
  |=============================================================================================================================                                                                                   |  60%
  |                                                                                                                                                                                                                      
  |==============================================================================================================================                                                                                  |  60%
  |                                                                                                                                                                                                                      
  |==============================================================================================================================                                                                                  |  61%
  |                                                                                                                                                                                                                      
  |===============================================================================================================================                                                                                 |  61%
  |                                                                                                                                                                                                                      
  |================================================================================================================================                                                                                |  61%
  |                                                                                                                                                                                                                      
  |================================================================================================================================                                                                                |  62%
  |                                                                                                                                                                                                                      
  |=================================================================================================================================                                                                               |  62%
  |                                                                                                                                                                                                                      
  |==================================================================================================================================                                                                              |  62%
  |                                                                                                                                                                                                                      
  |==================================================================================================================================                                                                              |  63%
  |                                                                                                                                                                                                                      
  |===================================================================================================================================                                                                             |  63%
  |                                                                                                                                                                                                                      
  |====================================================================================================================================                                                                            |  63%
  |                                                                                                                                                                                                                      
  |====================================================================================================================================                                                                            |  64%
  |                                                                                                                                                                                                                      
  |=====================================================================================================================================                                                                           |  64%
  |                                                                                                                                                                                                                      
  |======================================================================================================================================                                                                          |  64%
  |                                                                                                                                                                                                                      
  |======================================================================================================================================                                                                          |  65%
  |                                                                                                                                                                                                                      
  |=======================================================================================================================================                                                                         |  65%
  |                                                                                                                                                                                                                      
  |========================================================================================================================================                                                                        |  65%
  |                                                                                                                                                                                                                      
  |========================================================================================================================================                                                                        |  66%
  |                                                                                                                                                                                                                      
  |=========================================================================================================================================                                                                       |  66%
  |                                                                                                                                                                                                                      
  |==========================================================================================================================================                                                                      |  66%
  |                                                                                                                                                                                                                      
  |==========================================================================================================================================                                                                      |  67%
  |                                                                                                                                                                                                                      
  |===========================================================================================================================================                                                                     |  67%
  |                                                                                                                                                                                                                      
  |============================================================================================================================================                                                                    |  67%
  |                                                                                                                                                                                                                      
  |============================================================================================================================================                                                                    |  68%
  |                                                                                                                                                                                                                      
  |=============================================================================================================================================                                                                   |  68%
  |                                                                                                                                                                                                                      
  |==============================================================================================================================================                                                                  |  68%
  |                                                                                                                                                                                                                      
  |==============================================================================================================================================                                                                  |  69%
  |                                                                                                                                                                                                                      
  |===============================================================================================================================================                                                                 |  69%
  |                                                                                                                                                                                                                      
  |================================================================================================================================================                                                                |  69%
  |                                                                                                                                                                                                                      
  |=================================================================================================================================================                                                               |  69%
  |                                                                                                                                                                                                                      
  |=================================================================================================================================================                                                               |  70%
  |                                                                                                                                                                                                                      
  |==================================================================================================================================================                                                              |  70%
  |                                                                                                                                                                                                                      
  |===================================================================================================================================================                                                             |  70%
  |                                                                                                                                                                                                                      
  |===================================================================================================================================================                                                             |  71%
  |                                                                                                                                                                                                                      
  |====================================================================================================================================================                                                            |  71%
  |                                                                                                                                                                                                                      
  |=====================================================================================================================================================                                                           |  71%
  |                                                                                                                                                                                                                      
  |=====================================================================================================================================================                                                           |  72%
  |                                                                                                                                                                                                                      
  |======================================================================================================================================================                                                          |  72%
  |                                                                                                                                                                                                                      
  |=======================================================================================================================================================                                                         |  72%
  |                                                                                                                                                                                                                      
  |=======================================================================================================================================================                                                         |  73%
  |                                                                                                                                                                                                                      
  |========================================================================================================================================================                                                        |  73%
  |                                                                                                                                                                                                                      
  |=========================================================================================================================================================                                                       |  73%
  |                                                                                                                                                                                                                      
  |=========================================================================================================================================================                                                       |  74%
  |                                                                                                                                                                                                                      
  |==========================================================================================================================================================                                                      |  74%
  |                                                                                                                                                                                                                      
  |===========================================================================================================================================================                                                     |  74%
  |                                                                                                                                                                                                                      
  |===========================================================================================================================================================                                                     |  75%
  |                                                                                                                                                                                                                      
  |============================================================================================================================================================                                                    |  75%
  |                                                                                                                                                                                                                      
  |=============================================================================================================================================================                                                   |  75%
  |                                                                                                                                                                                                                      
  |=============================================================================================================================================================                                                   |  76%
  |                                                                                                                                                                                                                      
  |==============================================================================================================================================================                                                  |  76%
  |                                                                                                                                                                                                                      
  |===============================================================================================================================================================                                                 |  76%
  |                                                                                                                                                                                                                      
  |===============================================================================================================================================================                                                 |  77%
  |                                                                                                                                                                                                                      
  |================================================================================================================================================================                                                |  77%
  |                                                                                                                                                                                                                      
  |=================================================================================================================================================================                                               |  77%
  |                                                                                                                                                                                                                      
  |=================================================================================================================================================================                                               |  78%
  |                                                                                                                                                                                                                      
  |==================================================================================================================================================================                                              |  78%
  |                                                                                                                                                                                                                      
  |===================================================================================================================================================================                                             |  78%
  |                                                                                                                                                                                                                      
  |===================================================================================================================================================================                                             |  79%
  |                                                                                                                                                                                                                      
  |====================================================================================================================================================================                                            |  79%
  |                                                                                                                                                                                                                      
  |=====================================================================================================================================================================                                           |  79%
  |                                                                                                                                                                                                                      
  |=====================================================================================================================================================================                                           |  80%
  |                                                                                                                                                                                                                      
  |======================================================================================================================================================================                                          |  80%
  |                                                                                                                                                                                                                      
  |=======================================================================================================================================================================                                         |  80%
  |                                                                                                                                                                                                                      
  |=======================================================================================================================================================================                                         |  81%
  |                                                                                                                                                                                                                      
  |========================================================================================================================================================================                                        |  81%
  |                                                                                                                                                                                                                      
  |=========================================================================================================================================================================                                       |  81%
  |                                                                                                                                                                                                                      
  |==========================================================================================================================================================================                                      |  81%
  |                                                                                                                                                                                                                      
  |==========================================================================================================================================================================                                      |  82%
  |                                                                                                                                                                                                                      
  |===========================================================================================================================================================================                                     |  82%
  |                                                                                                                                                                                                                      
  |============================================================================================================================================================================                                    |  82%
  |                                                                                                                                                                                                                      
  |============================================================================================================================================================================                                    |  83%
  |                                                                                                                                                                                                                      
  |=============================================================================================================================================================================                                   |  83%
  |                                                                                                                                                                                                                      
  |==============================================================================================================================================================================                                  |  83%
  |                                                                                                                                                                                                                      
  |==============================================================================================================================================================================                                  |  84%
  |                                                                                                                                                                                                                      
  |===============================================================================================================================================================================                                 |  84%
  |                                                                                                                                                                                                                      
  |================================================================================================================================================================================                                |  84%
  |                                                                                                                                                                                                                      
  |================================================================================================================================================================================                                |  85%
  |                                                                                                                                                                                                                      
  |=================================================================================================================================================================================                               |  85%
  |                                                                                                                                                                                                                      
  |==================================================================================================================================================================================                              |  85%
  |                                                                                                                                                                                                                      
  |==================================================================================================================================================================================                              |  86%
  |                                                                                                                                                                                                                      
  |===================================================================================================================================================================================                             |  86%
  |                                                                                                                                                                                                                      
  |====================================================================================================================================================================================                            |  86%
  |                                                                                                                                                                                                                      
  |====================================================================================================================================================================================                            |  87%
  |                                                                                                                                                                                                                      
  |=====================================================================================================================================================================================                           |  87%
  |                                                                                                                                                                                                                      
  |======================================================================================================================================================================================                          |  87%
  |                                                                                                                                                                                                                      
  |======================================================================================================================================================================================                          |  88%
  |                                                                                                                                                                                                                      
  |=======================================================================================================================================================================================                         |  88%
  |                                                                                                                                                                                                                      
  |========================================================================================================================================================================================                        |  88%
  |                                                                                                                                                                                                                      
  |========================================================================================================================================================================================                        |  89%
  |                                                                                                                                                                                                                      
  |=========================================================================================================================================================================================                       |  89%
  |                                                                                                                                                                                                                      
  |==========================================================================================================================================================================================                      |  89%
  |                                                                                                                                                                                                                      
  |==========================================================================================================================================================================================                      |  90%
  |                                                                                                                                                                                                                      
  |===========================================================================================================================================================================================                     |  90%
  |                                                                                                                                                                                                                      
  |============================================================================================================================================================================================                    |  90%
  |                                                                                                                                                                                                                      
  |============================================================================================================================================================================================                    |  91%
  |                                                                                                                                                                                                                      
  |=============================================================================================================================================================================================                   |  91%
  |                                                                                                                                                                                                                      
  |==============================================================================================================================================================================================                  |  91%
  |                                                                                                                                                                                                                      
  |==============================================================================================================================================================================================                  |  92%
  |                                                                                                                                                                                                                      
  |===============================================================================================================================================================================================                 |  92%
  |                                                                                                                                                                                                                      
  |================================================================================================================================================================================================                |  92%
  |                                                                                                                                                                                                                      
  |================================================================================================================================================================================================                |  93%
  |                                                                                                                                                                                                                      
  |=================================================================================================================================================================================================               |  93%
  |                                                                                                                                                                                                                      
  |==================================================================================================================================================================================================              |  93%
  |                                                                                                                                                                                                                      
  |==================================================================================================================================================================================================              |  94%
  |                                                                                                                                                                                                                      
  |===================================================================================================================================================================================================             |  94%
  |                                                                                                                                                                                                                      
  |====================================================================================================================================================================================================            |  94%
  |                                                                                                                                                                                                                      
  |=====================================================================================================================================================================================================           |  94%
  |                                                                                                                                                                                                                      
  |=====================================================================================================================================================================================================           |  95%
  |                                                                                                                                                                                                                      
  |======================================================================================================================================================================================================          |  95%
  |                                                                                                                                                                                                                      
  |=======================================================================================================================================================================================================         |  95%
  |                                                                                                                                                                                                                      
  |=======================================================================================================================================================================================================         |  96%
  |                                                                                                                                                                                                                      
  |========================================================================================================================================================================================================        |  96%
  |                                                                                                                                                                                                                      
  |=========================================================================================================================================================================================================       |  96%
  |                                                                                                                                                                                                                      
  |=========================================================================================================================================================================================================       |  97%
  |                                                                                                                                                                                                                      
  |==========================================================================================================================================================================================================      |  97%
  |                                                                                                                                                                                                                      
  |===========================================================================================================================================================================================================     |  97%
  |                                                                                                                                                                                                                      
  |===========================================================================================================================================================================================================     |  98%
  |                                                                                                                                                                                                                      
  |============================================================================================================================================================================================================    |  98%
  |                                                                                                                                                                                                                      
  |=============================================================================================================================================================================================================   |  98%
  |                                                                                                                                                                                                                      
  |=============================================================================================================================================================================================================   |  99%
  |                                                                                                                                                                                                                      
  |==============================================================================================================================================================================================================  |  99%
  |                                                                                                                                                                                                                      
  |=============================================================================================================================================================================================================== |  99%
  |                                                                                                                                                                                                                      
  |=============================================================================================================================================================================================================== | 100%
  |                                                                                                                                                                                                                      
  |================================================================================================================================================================================================================| 100%
Centering and scaling data matrix

  |                                                                                                                                                                                                                      
  |                                                                                                                                                                                                                |   0%
  |                                                                                                                                                                                                                      
  |============                                                                                                                                                                                                    |   6%
  |                                                                                                                                                                                                                      
  |========================                                                                                                                                                                                        |  12%
  |                                                                                                                                                                                                                      
  |=====================================                                                                                                                                                                           |  18%
  |                                                                                                                                                                                                                      
  |=================================================                                                                                                                                                               |  24%
  |                                                                                                                                                                                                                      
  |=============================================================                                                                                                                                                   |  29%
  |                                                                                                                                                                                                                      
  |=========================================================================                                                                                                                                       |  35%
  |                                                                                                                                                                                                                      
  |======================================================================================                                                                                                                          |  41%
  |                                                                                                                                                                                                                      
  |==================================================================================================                                                                                                              |  47%
  |                                                                                                                                                                                                                      
  |==============================================================================================================                                                                                                  |  53%
  |                                                                                                                                                                                                                      
  |==========================================================================================================================                                                                                      |  59%
  |                                                                                                                                                                                                                      
  |=======================================================================================================================================                                                                         |  65%
  |                                                                                                                                                                                                                      
  |===================================================================================================================================================                                                             |  71%
  |                                                                                                                                                                                                                      
  |===============================================================================================================================================================                                                 |  76%
  |                                                                                                                                                                                                                      
  |===========================================================================================================================================================================                                     |  82%
  |                                                                                                                                                                                                                      
  |========================================================================================================================================================================================                        |  88%
  |                                                                                                                                                                                                                      
  |====================================================================================================================================================================================================            |  94%
  |                                                                                                                                                                                                                      
  |================================================================================================================================================================================================================| 100%

Save raw object

saveRDS(cmp.object, file = paste0(projectName, "_raw.RDS"))
cmp.object <- RunPCA(cmp.object, features = VariableFeatures(cmp.object), ndims.print = 1:5, nfeatures.print = 5)
PC_ 1 
Positive:  Vamp5, Nkg7, Car2, Apoe, Ctla2a 
Negative:  Lgals3, Aif1, Id2, Cst3, H2-Aa 
PC_ 2 
Positive:  Prtn3, Ctsg, H2afy, Mpo, Emb 
Negative:  Ube2c, Cenpf, Nusap1, Mki67, Cenpa 
PC_ 3 
Positive:  Pf4, Tmsb4x, Cavin2, Serpine2, Rap1b 
Negative:  Plac8, Cks2, Cenpa, Ube2c, Tubb4b 
PC_ 4 
Positive:  Csrp3, Car1, Jun, Apoe, Jund 
Negative:  H2afz, Hmgn2, Birc5, Hmgb1, Tuba1b 
PC_ 5 
Positive:  Pclaf, Tyms, Rrm2, Tk1, Pcna 
Negative:  Hist1h2bc, Tsc22d1, Smim14, Ccnb2, Serpinb1a 
DimPlot(cmp.object, reduction = "pca", group.by = "orig.ident")

VizDimLoadings(cmp.object, dims = 1:6, nfeatures = 10, reduction = "pca", ncol = 3)

Calculate dimensionality

ElbowPlot(cmp.object, ndims = 50)
percent.variance(cmp.object@reductions$pca@stdev)

Number of PCs describing X% of variance

ElbowPlot(cmp.object, ndims = 50)

percent.variance(cmp.object@reductions$pca@stdev)

Add cluster IDs from Seurat v1

Exported cell IDs for clusters 3, 17, 10, 11 from Seurat v1. Will add these IDs as a metadata column.
Create column “clust.ID” and populate with 0’s. Then import IDs for clusters

tot.var <- percent.variance(cmp.object@reductions$pca@stdev, plot.var = FALSE, return.val = TRUE)
paste0("Num pcs for 80% variance:", length(which(cumsum(tot.var) <= 80)))
[1] "Num pcs for 80% variance:18"
paste0("Num pcs for 85% variance:", length(which(cumsum(tot.var) <= 85)))
[1] "Num pcs for 85% variance:25"
paste0("Num pcs for 90% variance:", length(which(cumsum(tot.var) <= 90)))
[1] "Num pcs for 90% variance:33"
paste0("Num pcs for 95% variance:", length(which(cumsum(tot.var) <= 95)))
[1] "Num pcs for 95% variance:41"

Add new metadata column

clust3.cells <- read.table(file = "../Seuratv1_clusterCellIDs/cluster3cellIDs.txt", col.names = "clust03")
clust3.cells <- sapply(clust3.cells, function(x) paste0(gsub("CMP", "CMPm2", x), "-1"))
clust17.cells <- read.table(file = "../Seuratv1_clusterCellIDs/cluster17cellIDs.txt", col.names = "clust17")
clust17.cells <- sapply(clust17.cells, function(x) paste0(gsub("CMP", "CMPm2", x), "-1"))
clust10.cells <- read.table(file = "../Seuratv1_clusterCellIDs/cluster10cellIDs.txt", col.names = "clust10")
clust10.cells <- sapply(clust10.cells, function(x) paste0(gsub("CMP", "CMPm2", x), "-1"))
clust11.cells <- read.table(file = "../Seuratv1_clusterCellIDs/cluster11cellIDs.txt", col.names = "clust11")
clust11.cells <- sapply(clust11.cells, function(x) paste0(gsub("CMP", "CMPm2", x), "-1"))

now map new ids

cmp.object@meta.data['clust.ID'] <- 0
head(cmp.object@meta.data)
Registered S3 method overwritten by 'cli':
  method     from         
  print.boxx spatstat.geom

do numbers make sense?

cmp.object@meta.data$clust.ID[rownames(cmp.object@meta.data) %in% clust3.cells] <- 3
cmp.object@meta.data$clust.ID[rownames(cmp.object@meta.data) %in% clust17.cells] <- 17
cmp.object@meta.data$clust.ID[rownames(cmp.object@meta.data) %in% clust10.cells] <- 10
cmp.object@meta.data$clust.ID[rownames(cmp.object@meta.data) %in% clust11.cells] <- 11

Identify variable genes for new biomark

nrow(cmp.object@meta.data[cmp.object@meta.data$clust.ID == 10,])
[1] 1049
nrow(cmp.object@meta.data[cmp.object@meta.data$clust.ID == 11,])
[1] 1118
nrow(cmp.object@meta.data[cmp.object@meta.data$clust.ID == 17,])
[1] 883
nrow(cmp.object@meta.data[cmp.object@meta.data$clust.ID == 3,])
[1] 1931
length(cmp.object@assays$RNA@var.features)
[1] 2000

Total var 90%

Neighborhood and umap

set total.var <- 90%

top100.var <- cmp.object@assays$RNA@var.features[1:100]
length(top100.var)
[1] 100

Plot UMAP

tot.var <- percent.variance(cmp.object@reductions$pca@stdev, plot.var = FALSE, return.val = TRUE)
ndims <- length(which(cumsum(tot.var) <= 90))

cmp.object <- FindNeighbors(cmp.object, dims = 1:ndims)
Computing nearest neighbor graph
Computing SNN
cmp.object <- FindClusters(cmp.object, resolution = 0.5)
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 452999

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.8502
Number of communities: 9
Elapsed time: 1 seconds
cmp.object <- RunUMAP(cmp.object, dims = 1: ndims)
Warning: The default method for RunUMAP has changed from calling Python UMAP via reticulate to the R-native UWOT using the cosine metric
To use Python UMAP via reticulate, set umap.method to 'umap-learn' and metric to 'correlation'
This message will be shown once per session
13:50:56 UMAP embedding parameters a = 0.9922 b = 1.112
13:50:56 Read 12059 rows and found 33 numeric columns
13:50:56 Using Annoy for neighbor search, n_neighbors = 30
13:50:56 Building Annoy index with metric = cosine, n_trees = 50
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
13:50:57 Writing NN index file to temp file /var/folders/4f/fwrj6fnn1dn4g8wsf0zv563hjsvl24/T//Rtmp5gGDJN/file582a292245c6
13:50:57 Searching Annoy index using 1 thread, search_k = 3000
13:50:59 Annoy recall = 100%
13:51:00 Commencing smooth kNN distance calibration using 1 thread
13:51:00 Initializing from normalized Laplacian + noise
13:51:01 Commencing optimization for 200 epochs, with 513294 positive edges
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
13:51:06 Optimization finished
for(x in c(0.5, 1, 1.5, 2, 2.5)){
    cmp.object <- FindClusters(cmp.object, resolution = x)
}
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 452999

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.8502
Number of communities: 9
Elapsed time: 1 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 452999

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.7974
Number of communities: 17
Elapsed time: 1 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 452999

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.7652
Number of communities: 22
Elapsed time: 1 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 452999

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.7404
Number of communities: 27
Elapsed time: 1 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 452999

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.7189
Number of communities: 30
Elapsed time: 1 seconds
saveRDS(cmp.object, file = paste0(projectName, "_dim", ndims, ".RDS"))

Clustree

what’s the max resolution we can achieve while keepign clusters stable?

for (meta.col in colnames(cmp.object@meta.data)){
    if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
        myplot <- DimPlot(cmp.object, 
                                            group.by = meta.col,
                                            reduction = "umap", 
                                            cols = color.palette
                                            ) + 
            ggtitle(paste0(projectName, " dim", ndims, "res", gsub("RNA_snn_res", "", meta.col) ))
        plot(myplot)
    }
}
Error in DimPlot(cmp.object, group.by = meta.col, reduction = "umap",  : 
  object 'color.palette' not found

I think I’m liking res.1.0 from this. Although how much does this change if I use fewer PCs…

for each resolution, number/percentage of cells in each cluster?

plot.title <- paste0(projectName, "_clustree_ndim", max(cmp.object@commands$RunUMAP.RNA.pca$dims))
my.clustree <- clustree(cmp.object, prefix = "RNA_snn_res.", node_colour = "sc3_stability", exprs = "scale.data") + 
    scale_color_continuous(low = 'red3', high = 'white') + 
    ggtitle(plot.title)
Error: Less than two column names matched the prefix: RNA_snn_res.

For each resolution, what percentage of cells in each cluster are enriched for one of our clust.IDs?

Test: what percentage of each new clusterID matches one of the older clusters?

tot.cells <- nrow(cmp.object@meta.data)
for (meta.col in colnames(cmp.object@meta.data)){
    if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
        new.clusters <- sort(as.numeric(levels(cmp.object@meta.data[[meta.col]])))
        stats.df <- data.frame(matrix(ncol = 2, nrow = length(new.clusters)))
        colnames(stats.df) <- c("num_cells", "pct_pop")
        rownames(stats.df) <- new.clusters
        meta.df <- cmp.object@meta.data
        for(row.id in rownames(stats.df)){
                num.x <- nrow(meta.df[meta.df[meta.col] == row.id,])
                pct.x <- as.integer(num.x / tot.cells *100)
                # print(pct.x)
                stats.df[row.id, "num_cells"] <- num.x
                stats.df[row.id, "pct_pop"] <- pct.x
        }
        print(stats.df)
    }
}

Absolutely terrible overlap, no enrichment of any of these across the new clustering algorithm. Maybe should try 95% variation covered

Find old cells on UMAP

time for the super scarey moment to see if the cells from seuratv1 still cluster together on in seurat v4

for (meta.col in colnames(cmp.object@meta.data)){
    if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
        new.clusters <- sort(as.numeric(levels(cmp.object@meta.data[[meta.col]])))
        enrich.df <- data.frame(matrix(ncol = 4, nrow = length(new.clusters)))
        colnames(enrich.df) <- c(3, 17, 10, 11)
        rownames(enrich.df) <- new.clusters
        meta.df <- cmp.object@meta.data
        for(row.id in rownames(enrich.df)){
            tot.clus <- nrow(meta.df[meta.df[[meta.col]] == row.id,])
            for(col.id in colnames(enrich.df)){
                num.x <- nrow(meta.df[(meta.df[[meta.col]] == row.id) & (meta.df$clust.ID == col.id),])
                pct.x <- as.integer(num.x / tot.clus *100)
                # print(pct.x)
                enrich.df[row.id, col.id] <- pct.x
            }
        }
        colnames(enrich.df) <- sapply(colnames(enrich.df), function(x) paste0("oldcluster", x))
        rownames(enrich.df) <- sapply(rownames(enrich.df), function(x) paste0("newcluster", x))
        xlsx::write.xlsx(enrich.df, file = paste0("PctOfNewClustersOverlappingOldClusters_", projectName, "_dim", ndims, ".xlsx"), sheetName = paste0(gsub("RNA_snn_", "", meta.col)), append = TRUE)
        print(enrich.df)
    }
}
NA
DimPlot(cmp.object,
                reduction = "umap",
                group.by = "clust.ID", 
                # split.by = "orig.ident",
                cols = c("gray", "orange", "blue", "red", "green"),)

Total var 85%

Neighborhood and umap

set total.var <- 85%

DimPlot(cmp.object,
                reduction = "umap",
                group.by = "orig.ident", 
                split.by = "clust.ID",
                cols = c("gray", "orange", "blue", "red", "green"),)

Plot UMAP

tot.var <- percent.variance(cmp.object@reductions$pca@stdev, plot.var = FALSE, return.val = TRUE)
ndims <- length(which(cumsum(tot.var) <= 85))

cmp.object <- FindNeighbors(cmp.object, dims = 1:ndims)
Computing nearest neighbor graph
Computing SNN
cmp.object <- FindClusters(cmp.object, resolution = 0.5)
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 424091

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.8508
Number of communities: 9
Elapsed time: 2 seconds
cmp.object <- RunUMAP(cmp.object, dims = 1: ndims)
Warning: The default method for RunUMAP has changed from calling Python UMAP via reticulate to the R-native UWOT using the cosine metric
To use Python UMAP via reticulate, set umap.method to 'umap-learn' and metric to 'correlation'
This message will be shown once per session
11:05:25 UMAP embedding parameters a = 0.9922 b = 1.112
11:05:25 Read 12059 rows and found 25 numeric columns
11:05:25 Using Annoy for neighbor search, n_neighbors = 30
11:05:25 Building Annoy index with metric = cosine, n_trees = 50
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
11:05:26 Writing NN index file to temp file /var/folders/4f/fwrj6fnn1dn4g8wsf0zv563hjsvl24/T//RtmpN5aoAI/file18065624a624
11:05:26 Searching Annoy index using 1 thread, search_k = 3000
11:05:30 Annoy recall = 100%
11:05:30 Commencing smooth kNN distance calibration using 1 thread
11:05:31 Initializing from normalized Laplacian + noise
11:05:31 Commencing optimization for 200 epochs, with 500658 positive edges
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
11:05:38 Optimization finished
saveRDS(cmp.object, file = paste0(projectName, "_dim", ndims, ".RDS"))
for(x in c(0.5, 1, 1.5, 2, 2.5)){
    cmp.object <- FindClusters(cmp.object, resolution = x)
}
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 424091

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.8508
Number of communities: 9
Elapsed time: 2 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 424091

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.8026
Number of communities: 18
Elapsed time: 1 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 424091

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.7700
Number of communities: 22
Elapsed time: 1 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 424091

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.7461
Number of communities: 28
Elapsed time: 1 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 424091

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.7257
Number of communities: 33
Elapsed time: 1 seconds

Clustree

what’s the max resolution we can achieve while keepign clusters stable?

for (meta.col in colnames(cmp.object@meta.data)){
    if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
        myplot <- DimPlot(cmp.object, 
                                            group.by = meta.col,
                                            reduction = "umap", 
                                            cols = color.palette
                                            ) + 
            ggtitle(paste0(projectName, " dim", ndims, "res", gsub("RNA_snn_res", "", meta.col) ))
        plot(myplot)
    }
}

for each resolution, number/percentage of cells in each cluster?

tot.cells <- nrow(cmp.object@meta.data)
for (meta.col in colnames(cmp.object@meta.data)){
    if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
        new.clusters <- sort(as.numeric(levels(cmp.object@meta.data[[meta.col]])))
        stats.df <- data.frame(matrix(ncol = 2, nrow = length(new.clusters)))
        colnames(stats.df) <- c("num_cells", "pct_pop")
        rownames(stats.df) <- new.clusters
        meta.df <- cmp.object@meta.data
        for(row.id in rownames(stats.df)){
                num.x <- nrow(meta.df[meta.df[meta.col] == row.id,])
                pct.x <- as.integer(num.x / tot.cells *100)
                # print(pct.x)
                stats.df[row.id, "num_cells"] <- num.x
                stats.df[row.id, "pct_pop"] <- pct.x
        }
        print(stats.df)
    }
}

For each resolution, what percentage of cells in each cluster are enriched for one of our clust.IDs?

Test: what percentage of each new clusterID matches one of the older clusters?

for (meta.col in colnames(cmp.object@meta.data)){
    if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
        new.clusters <- sort(as.numeric(levels(cmp.object@meta.data[[meta.col]])))
        enrich.df <- data.frame(matrix(ncol = 4, nrow = length(new.clusters)))
        colnames(enrich.df) <- c(3, 17, 10, 11)
        rownames(enrich.df) <- new.clusters
        meta.df <- cmp.object@meta.data
        for(row.id in rownames(enrich.df)){
            tot.clus <- nrow(meta.df[meta.df[[meta.col]] == row.id,])
            for(col.id in colnames(enrich.df)){
                num.x <- nrow(meta.df[(meta.df[[meta.col]] == row.id) & (meta.df$clust.ID == col.id),])
                pct.x <- as.integer(num.x / tot.clus *100)
                # print(pct.x)
                enrich.df[row.id, col.id] <- pct.x
            }
        }
        colnames(enrich.df) <- sapply(colnames(enrich.df), function(x) paste0("oldcluster", x))
        rownames(enrich.df) <- sapply(rownames(enrich.df), function(x) paste0("newcluster", x))
        xlsx::write.xlsx(enrich.df, file = paste0("PctOfNewClustersOverlappingOldClusters_", projectName, "_dim", ndims, ".xlsx"), sheetName = paste0(gsub("RNA_snn_", "", meta.col)), append = TRUE)
        print(enrich.df)
    }
}

Absolutely terrible overlap, no enrichment of any of these across the new clustering algorithm. Maybe should try 95% variation covered

Find old cells on UMAP

time for the super scarey moment to see if the cells from seuratv1 still cluster together on in seurat v4

DimPlot(cmp.object,
                reduction = "umap",
                group.by = "clust.ID", 
                # split.by = "orig.ident",
                cols = c("gray", "orange", "blue", "red", "green"),)
DimPlot(cmp.object,
                reduction = "umap",
                group.by = "orig.ident", 
                split.by = "clust.ID",
                cols = c("gray", "orange", "blue", "red", "green"),)

Total var 95%

Neighborhood and umap

set total.var <- 95%

tot.var <- percent.variance(cmp.object@reductions$pca@stdev, plot.var = FALSE, return.val = TRUE)
ndims <- length(which(cumsum(tot.var) <= 95))

cmp.object <- FindNeighbors(cmp.object, dims = 1:ndims)
cmp.object <- FindClusters(cmp.object, resolution = 0.5)
cmp.object <- RunUMAP(cmp.object, dims = 1: ndims)

saveRDS(cmp.object, file = paste0(projectName, "_dim", ndims, ".RDS"))

Plot UMAP

tot.var <- percent.variance(cmp.object@reductions$pca@stdev, plot.var = FALSE, return.val = TRUE)
ndims <- length(which(cumsum(tot.var) <= 95))

cmp.object <- FindNeighbors(cmp.object, dims = 1:ndims)
Computing nearest neighbor graph
Computing SNN
cmp.object <- FindClusters(cmp.object, resolution = 0.5)
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 478535

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.8472
Number of communities: 9
Elapsed time: 1 seconds
cmp.object <- RunUMAP(cmp.object, dims = 1: ndims)
07:23:01 UMAP embedding parameters a = 0.9922 b = 1.112
07:23:01 Read 12059 rows and found 41 numeric columns
07:23:01 Using Annoy for neighbor search, n_neighbors = 30
07:23:01 Building Annoy index with metric = cosine, n_trees = 50
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
07:23:02 Writing NN index file to temp file /var/folders/4f/fwrj6fnn1dn4g8wsf0zv563hjsvl24/T//Rtmp6Y3Oqu/file12bf353fef34f
07:23:02 Searching Annoy index using 1 thread, search_k = 3000
07:23:06 Annoy recall = 100%
07:23:06 Commencing smooth kNN distance calibration using 1 thread
07:23:07 Initializing from normalized Laplacian + noise
07:23:08 Commencing optimization for 200 epochs, with 524686 positive edges
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
07:23:15 Optimization finished
saveRDS(cmp.object, file = paste0(projectName, "_dim", ndims, ".RDS"))
for(x in c(0.5, 1, 1.5, 2, 2.5)){
    cmp.object <- FindClusters(cmp.object, resolution = x)
}
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 478535

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.8472
Number of communities: 9
Elapsed time: 2 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 478535

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.7908
Number of communities: 17
Elapsed time: 1 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 478535

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.7591
Number of communities: 20
Elapsed time: 2 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 478535

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.7332
Number of communities: 26
Elapsed time: 1 seconds
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 12059
Number of edges: 478535

Running Louvain algorithm...
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Maximum modularity in 10 random starts: 0.7121
Number of communities: 32
Elapsed time: 1 seconds
cmp.object <- readRDS("CMP_dim41.RDS")

For each resolution, what percentage of cells in each cluster are enriched for one of our clust.IDs?

Test: what percentage of each new clusterID matches one of the older clusters?

for (meta.col in colnames(cmp.object@meta.data)){
    if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
        new.clusters <- sort(as.numeric(levels(cmp.object@meta.data[[meta.col]])))
        enrich.df <- data.frame(matrix(ncol = 4, nrow = length(new.clusters)))
        colnames(enrich.df) <- c(3, 17, 10, 11)
        rownames(enrich.df) <- new.clusters
        meta.df <- cmp.object@meta.data
        for(row.id in rownames(enrich.df)){
            tot.clus <- nrow(meta.df[meta.df[[meta.col]] == row.id,])
            for(col.id in colnames(enrich.df)){
                num.x <- nrow(meta.df[(meta.df[[meta.col]] == row.id) & (meta.df$clust.ID == col.id),])
                pct.x <- as.integer(num.x / tot.clus *100)
                # print(pct.x)
                enrich.df[row.id, col.id] <- pct.x
            }
        }
        colnames(enrich.df) <- sapply(colnames(enrich.df), function(x) paste0("oldcluster", x))
        rownames(enrich.df) <- sapply(rownames(enrich.df), function(x) paste0("newcluster", x))
        xlsx::write.xlsx(enrich.df, file = paste0("PctOfNewClustersOverlappingOldClusters_", projectName, "_dim", ndims, ".xlsx"), sheetName = paste0(gsub("RNA_snn_", "", meta.col)), append = TRUE)
        print(enrich.df)
    }
}

Absolutely terrible overlap, no enrichment of any of these across the new clustering algorithm. Maybe should try 95% variation covered

Find old cells on UMAP

time for the super scarey moment to see if the cells from seuratv1 still cluster together on in seurat v4

DimPlot(cmp.object,
                reduction = "umap",
                group.by = "clust.ID", 
                pt.size = .1,
                # split.by = "orig.ident",
                cols = c("gray", "orange", "blue", "red", "green"),)
DimPlot(cmp.object,
                reduction = "umap",
                group.by = "orig.ident", 
                split.by = "clust.ID",
                cols = c("gray", "orange", "blue", "red", "green"),)

Gene expression of old clustrs on new map

Let’s see if we can get some gene expression profiles on these…

gene.list <- c("Gata1", "Gata2", "Pf4", "Dntt", "Mpo", "Meis1", "Irf8", "Elane", "Fli1", "Zfpm1")
VlnPlot(cmp.object, features = gene.list, group.by = "clust.ID", pt.size = 0.01, cols = c("gray", "orange", "blue", "red", "green"))

Used the exce doc to do some fancy conditional formatting. Old cluster 17 is pretty dispersed until you it resolution 2.5. Otherise, cells in old cluster 17 do not constitute more than 40% of any cells in the new clusters.
As far as I can see, the two approaches are to do DGEof new CMP w/ resolution = 2.5, AND/OR do DGe using older cluster IDs. Sure seems to make sense to do both…

DGE w/ resolution = 2.5

Strt with comparing all clusters against all other clusters Write out cluster info

calculate FindAllMarkers() for different idents and save to new file

ident.list <- c("RNA_snn_res.0.5", "RNA_snn_res.1", "RNA_snn_res.1.5", "RNA_snn_res.2", "RNA_snn_res.2.5", "clust.ID")
for(tested.ident in ident.list){
    Idents(cmp.object) <- tested.ident
    all.markers <- FindAllMarkers(cmp.object)
    xlsx::write.xlsx(x = all.markers[,c("avg_log2FC", "p_val_adj", "cluster", "gene")], 
                                     file = paste0(projectName, "_FindALLMarkers_res2.5.xlsx"), 
                                     sheetName = tested.ident, 
                                     col.names = TRUE, 
                                     row.names = FALSE, 
                                     append = TRUE)
}

Create FindAllMarkers() lists for GSEA

Idents(cmp.object) <- "RNA_snn_res.2.5"
res.2.5.allmarkers <- FindAllMarkers(cmp.object)

Map HGNC symbols

Mouse2HumanTable <- Mouse2Human(res.2.5.allmarkers$gene)

HGNC <- with(Mouse2HumanTable, Mouse2HumanTable$HGNC[match(res.2.5.allmarkers$gene, Mouse2HumanTable$MGI)])
head(res.2.5.allmarkers)
res.2.5.allmarkers$HGNC <- HGNC
tail(res.2.5.allmarkers)
sig.res.2.5 <- res.2.5.allmarkers[res.2.5.allmarkers$p_val_adj <= 0.05, ]
sig.res.2.5 <- sig.res.2.5[c("avg_log2FC", "HGNC", "cluster")]
sig.res.2.5 <- sig.res.2.5[!(sig.res.2.5$HGNC == "" | is.na(sig.res.2.5$HGNC)),] # GSEA will fail if there are any blanks or NAs in the table
sig.res.2.5 <- sig.res.2.5[]
for(cluster in unique(sig.res.2.5$cluster)){
    print(paste("writing cluster", cluster))
    new.table <- sig.res.2.5[sig.res.2.5$cluster == cluster, c("HGNC", "avg_log2FC")]
    new.table <- new.table[order(-new.table$avg_log2FC), ]
    write.table(new.table, file = paste0("RankList_res2.5_findAll_hgnc/res.2.5cluster", cluster, ".rnk"), quote = FALSE, row.names = FALSE, col.names = TRUE, sep = "\t", )
    
}

calculate FindMarkers() that distinguish each cluster (might overlab between clusters)

ident.list <- c("RNA_snn_res.0.5", "RNA_snn_res.1", "RNA_snn_res.1.5", "RNA_snn_res.2", "RNA_snn_res.2.5", "clust.ID")
for(tested.ident in ident.list){
    object.copy <- cmp.object
    Idents(object.copy) <- tested.ident
    print(paste("testing", tested.ident))
    for (cluster in sort(as.numeric(levels(object.copy@meta.data[[tested.ident]])))){
        print(paste("looking at cluster", cluster))
        cluster.markers <- FindMarkers(object.copy, ident.1 = cluster)
        try(
            xlsx::write.xlsx(x = cluster.markers[,c("avg_log2FC", "p_val_adj")], 
                                             file = paste0(projectName, "_FindMarkers_", gsub("RNA_snn_", "", tested.ident), ".xlsx"), 
                                             sheetName = paste0("clst", cluster), 
                                             col.names = TRUE, 
                                             row.names = TRUE, 
                                             append = TRUE)
        )   
    }
    remove(object.copy)
}
for (cluster in sort(as.numeric(levels(cmp.object@meta.data$RNA_snn_res.2.5)))){
    cluster.markers <- FindMarkers(cmp.object, ident.1 = cluster)
    xlsx::write.xlsx(x = cluster.markers[,c("avg_log2FC", "p_val_adj")], 
                                     file = paste0(projectName, "_FindMarkers_res2.5.xlsx"), 
                                     sheetName = paste0("clst", cluster), 
                                     col.names = TRUE, 
                                     row.names = TRUE, 
                                     append = TRUE)
}

Combine clusters that might represent old cluster ids

DGE w/ metadata against clust.ID against “0”

reset ident as “clust.ID” and rerun FindAllMarkers()

    Idents(cmp.object) <- "clust.ID"
    all.markers <- FindAllMarkers(cmp.object)
    xlsx::write.xlsx(x = all.markers[,c("avg_log2FC", "p_val_adj", "cluster", "gene")], 
                                     file = paste0(projectName, "_FindALLMarkers_clustID.xlsx"), 
                                     sheetName = "clustID", 
                                     col.names = TRUE, 
                                     row.names = FALSE, 
                                     append = TRUE)
# Idents(cmp.object) <- "clust.ID"
for (cluster in unique(cmp.object@meta.data$clust.ID)){
    print(cluster)
    cluster.markers <- FindMarkers(cmp.object, ident.1 = cluster)
    xlsx::write.xlsx(x = cluster.markers[,c("avg_log2FC", "p_val_adj")], 
                                     file = paste0(projectName, "_FindMarkers_clustID.xlsx"), 
                                     sheetName = paste0("oldclust", cluster), 
                                     col.names = TRUE, 
                                     row.names = TRUE, 
                                     append = TRUE)
}

Distinguishing features of clusters

Previously defined biomark genes based on PC contributions. Original list was based on all msAggr, but let’s see how CMP subset does?

VizDimLoadings(cmp.object, dims = 1:10, nfeatures = 30, reduction = "pca", ncol = 2)
pca.df <- cmp.object[["pca"]]
pca.df <- as.data.frame(as.matrix(slot(object = pca.df, name = "feature.loadings")))
print(cmp.object[["pca"]], dims = 2, nfeatures = 5)
rownames(pca.df[pca.df$PC_2 %in% sort(pca.df$PC_2, decreasing = TRUE)[1:5], ])
rownames(pca.df[pca.df$PC_2 %in% sort(pca.df$PC_2)[1:5], ])

now we can get a list of principal components!
first pull the list of oldAnalysis CMP top PC genes

cmp.biomark <- read.table(file = "/Users/heustonef/Desktop/CMPSubpops/BioMark/ProbePanels/CMP_PCTopGenes.txt", sep = "\t", header = TRUE)
biomark.cmptargets <- c()
for(df.col in 1:ncol(cmp.biomark)){
    biomark.cmptargets <- c(biomark.cmptargets, biomark[,df.col])
}
print(colnames(biomark))
print(paste("total gene count:", length(biomark.cmptargets)))

Now get the list of current pc gene trgets (oldAnalysis used ndim = 1:6, so we’ll start with that range)

pc.list <- c("PC_1", "PC_2", "PC_3", "PC_4", "PC_5", "PC_6")
pc.genes <- lapply(pc.list, function(x) rownames(pca.df[pca.df[[x]] %in% sort(pca.df[[x]], decreasing = TRUE)[1:30],])) #targeting roughly 180 genes like in biomark.cmptargets
pc.genes <- unique(unlist(pc.genes))
print(paste("total gene count:", length(pc.genes)))

Now compare the lists, I guess:

# setdiff(x,y) gives you things in x not in y. setdiff(y,x) gives you things in y not in x
setdiff(biomark.cmptargets, pc.genes)
# print(paste("\n length:", length(setdiff(biomark.cmptargets, pc.genes))))
writeLines(c("", "length:", length(setdiff(biomark.cmptargets, pc.genes))))

Umm, yeah that went kinda how I expected. Let’s do this again, but for the actual biomark gene lists.

biomark <- read.table(file = "/Users/heustonef/Desktop/CMPSubpops/BioMark/ProbePanels/BiomarkProbeList.txt", sep = "\t")
biomark <- biomark[,1]
setdiff(biomark, pc.genes)
writeLines(c("", "length:", length(setdiff(biomark, pc.genes))))

What if we increase the number of pcs but decrease the depth of each? This might cover more of biomark, which was originally developed using msAggr instead of only the CMP subset

pc.list <- c("PC_1", "PC_2", "PC_3", "PC_4", "PC_5", "PC_6", "PC_7", "PC_8", "PC_9", "PC_10")
pc.genes <- lapply(pc.list, function(x) rownames(pca.df[pca.df[[x]] %in% sort(pca.df[[x]], decreasing = TRUE)[1:20],]))
pc.genes <- unique(unlist(pc.genes))
print(paste("total gene count:", length(pc.genes)))
setdiff(biomark, pc.genes)
writeLines(c("", "length:", length(setdiff(biomark, pc.genes))))

For comparison, let’s just see how many of biomark.cmptargets were actually included in biomark

setdiff(biomark.cmptargets, biomark)
writeLines(c("", "length:", length(setdiff(biomark.cmptargets, pc.genes))))
length(biomark) - length(setdiff(biomark, biomark.cmptargets))
length(biomark) - length(setdiff(biomark, pc.genes))

So when you look at it like that, it’s not actually that far off.

What are the similarities?:

setdiff(setdiff(biomark, biomark.cmptargets), setdiff(biomark, pc.genes))

These are genes from the 97probes not in the old CMP set that are also not in the new CMP set. Other than Itga2b (which is a failed probe anyway), nothing screams. Also we’d have thrown Flt3 and Cd34 for in anyway because they’re requisite cell surface markers (also Flt3 surface marker is expensive but otherwise not noteworthy and not used in the current sorting strategy)

What about cell surface marker expression? * Cd34 * Cd16/32 * Cd9 * Cd41 * Cd48 * Sca1 (just throw that in for sh*&s and giggles)

surface.markers <- c("Cd34", "Fcgr3", "Fcgr2b", "Cd9", "Itga2b", "Cd48", "Ly6a")
FeaturePlot(cmp.object, features = surface.markers, pt.size = 1, split.by = "clust.ID", ncol = 1)

Save as png

png(filename = "FeaturePlot_CMP_surfaceMarkers_clustIDfacet.png", height = 1600, width = 1600)
FeaturePlot(cmp.object, features = surface.markers, pt.size = 1, split.by = "clust.ID", ncol = 1)
dev.off()

Compare @g hierarchcial clusteirng

Do clustering using biomark RNAs as input

# Read in BiomarkRNAs
biomark.rnas <- read.table('/Users/heustonef/Desktop/10XGenomicsData/BiomarkRNAs.txt')
biomark.rnas <- biomark.rnas$V1

use biomark RNAs to define dimensional reduction

cmp.object <- readRDS("CMP_raw.RDS")
cmp.object <- RunPCA(cmp.object, features = biomark.rnas, ndims.print = 1:5, , nfeatures.print = 5)
ElbowPlot(cmp.object, ndims = 50)

Now run the clustering

tot.var <- percent.variance(cmp.object@reductions$pca@stdev, plot.var = FALSE, return.val = TRUE)
ndims <- length(which(cumsum(tot.var) <= 90))

cmp.object <- FindNeighbors(cmp.object, dims = 1:ndims)
cmp.object <- FindClusters(cmp.object, resolution = 0.5)
cmp.object <- RunUMAP(cmp.object, dims = 1: ndims)

find the clusters

for(x in c(0.5, 1, 1.5, 2, 2.5)){
    cmp.object <- FindClusters(cmp.object, resolution = x)
}

Plot the umaps and cell cluster ids

for (meta.col in colnames(cmp.object@meta.data)){
    if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
        myplot <- DimPlot(cmp.object, 
                                            group.by = meta.col,
                                            reduction = "umap", 
                                            cols = color.palette
                                            ) + 
            ggtitle(paste0(projectName, " dim", ndims, "res", gsub("RNA_snn_res", "", meta.col) ))
        plot(myplot)
    }
}

Calculate anticipated number of cells you’ll find in each biomark cluster

Get # cells in each cluster

tot.cellcount <- nrow(cmp.object@meta.data)
res05.list <- sort(unique(cmp.object@meta.data$RNA_snn_res.0.5), decreasing = FALSE)
sapply(res05.list, 
             function(x){
                print(
                    paste(
                        "cluster", x, "=", 
                        nrow(cmp.object@meta.data[cmp.object@meta.data$RNA_snn_res.0.5 == x,]), 
                        "cells or", 
                        round(nrow(cmp.object@meta.data[cmp.object@meta.data$RNA_snn_res.0.5 == x,])/tot.cellcount*100, digits = 2), 
                        "% of total"
                    )
                )
             }
            )

So we did the dimensional reduction based on the biomark RNAs, then did our UMAP nearest neighbor clustering.

In the biomark hierarchcial clustering analysis I assayed 167 cells. The smallest cluster I detected had 3 cells, or 1.8% of total, and this is an uncomfortably small number of cells. Based on the UMAP calculations I would therefore expect to find 11 or 12 of the predicted 15 clusters. I found 12, and I don’t really like that last one, so 11 or 12. Since I did the hierarchcial clustering yesterday and did this math today, we can say it was independent of these results and therefore totally legit. Yay!!

---
title: "CMPSubset"
output: html_notebook
---

# Updates

## Update 2021.08.11
Most thorough way to readdress differences between original and current analyses is to jsut redo the biomark. To that end, will restart analysis of *just* CMPm2, excluding other datasets (i.e., LSK, MEP, GMP). Will also keep 


# Notebook setup

Creating new pipeline using seurat v4.0.2 available 2021.06.08

Load libraries required for Seuratv4

```{r setup}
library(dplyr)
library(Seurat)
library(patchwork)
library(ggplot2)
library(clustree)
```

# Set global variables
```{r}
projectName <- "CMP"
jackstraw.dim <- 40
```

store session info
```{r}
sessionInfo.filename <- paste0(projectName, "_sessionInfo.txt")
sink(sessionInfo.filename)
sessionInfo()
sink()
```


```{r}
source("../RFunctions/read_10XGenomics_data.R")
source("../RFunctions/PercentVariance.R")
source("../RFunctions/Mouse2Human_idconversion.R")
```


```{r warning=FALSE}
setwd("../../cellRanger/") # temporarily changing wd only works if you run the entire chunk at once
data_file.list <- read_10XGenomics_data(sample.list = "CMPm2")
object.data <-Read10X(data_file.list)
```



```{r}
cmp.object<- CreateSeuratObject(counts = object.data, min.cells = 3, min.genes = 200, project = projectName)
```

Clean up to free memory

```{r}
remove(object.data)
```


Add mitochondrial metadata and plot some basic features
```{r}
cmp.object[["percent.mt"]] <- PercentageFeatureSet(cmp.object, pattern = "^mt-")
VlnPlot(cmp.object, features = c("nFeature_RNA", "nCount_RNA", "percent.mt"), ncol = 3, pt.size = 0, fill.by = 'orig.ident', )
```


```{r}
plot1 <- FeatureScatter(cmp.object, feature1 = "nCount_RNA", feature2 = "percent.mt", group.by = "orig.ident", pt.size = 0.01)
plot2 <- FeatureScatter(cmp.object, feature1 = "nCount_RNA", feature2 = "nFeature_RNA", group.by = "orig.ident", pt.size = 0.01)
plot1 + plot2
```
## Filter data
remove low quality cells
require: nFeature_RNA between 200 and 4000 (inclusive)
require: percent.mt <=5

```{r}
print(paste("original object:", nrow(cmp.object@meta.data), "cells", sep = " "))
cmp.object <- subset(cmp.object, 
												subset = nFeature_RNA >=200 & 
													nFeature_RNA <= 4000 & 
													percent.mt <= 5
												)
print(paste("new object:", nrow(cmp.object@meta.data), "cells", sep = " "))
```



```{r}
cmp.object <- NormalizeData(cmp.object, normalization.method = "LogNormalize", scale.factor = 10000)
```


Find variable features
```{r fig.width = 5, fig.height = 2}
cmp.object <- FindVariableFeatures(cmp.object, selection.method = "vst", nfeatures = 2000)
top10 <- head(VariableFeatures(cmp.object), 10)
plot1 <- VariableFeaturePlot(cmp.object)
plot2 <- LabelPoints(plot = plot1, points = top10, repel = TRUE)
plot1 + plot2
```

## Scale data (linear transformation)
We don't have to worry about comparing library depths, so we'll just do normalization/Scale data

```{r echo = FALSE}
all.genes <- rownames(cmp.object)
cmp.object <- ScaleData(cmp.object, features = all.genes, vars.to.regress = c("nFeature_RNA", "nCount_RNA"))
```

## Save raw object
```{r}
saveRDS(cmp.object, file = paste0(projectName, "_raw.RDS"))
```



```{r}
cmp.object <- RunPCA(cmp.object, features = VariableFeatures(cmp.object), ndims.print = 1:5, nfeatures.print = 5)
```

```{r}
DimPlot(cmp.object, reduction = "pca", group.by = "orig.ident")
VizDimLoadings(cmp.object, dims = 1:6, nfeatures = 10, reduction = "pca", ncol = 3)

```

Calculate dimensionality
```{r, figures-side, fig.show='hold', out.width="50%"}
ElbowPlot(cmp.object, ndims = 50)
percent.variance(cmp.object@reductions$pca@stdev)
```
Number of PCs describing X% of variance

```{r}
tot.var <- percent.variance(cmp.object@reductions$pca@stdev, plot.var = FALSE, return.val = TRUE)
paste0("Num pcs for 80% variance:", length(which(cumsum(tot.var) <= 80)))
paste0("Num pcs for 85% variance:", length(which(cumsum(tot.var) <= 85)))
paste0("Num pcs for 90% variance:", length(which(cumsum(tot.var) <= 90)))
paste0("Num pcs for 95% variance:", length(which(cumsum(tot.var) <= 95)))

```

# Add cluster IDs from Seurat v1

Exported cell IDs for clusters 3, 17, 10, 11 from Seurat v1. Will add these IDs as a metadata column.  
Create column "clust.ID" and populate with 0's. Then import IDs for clusters



```{r}
clust3.cells <- read.table(file = "../Seuratv1_clusterCellIDs/cluster3cellIDs.txt", col.names = "clust03")
clust3.cells <- sapply(clust3.cells, function(x) paste0(gsub("CMP", "CMPm2", x), "-1"))
clust17.cells <- read.table(file = "../Seuratv1_clusterCellIDs/cluster17cellIDs.txt", col.names = "clust17")
clust17.cells <- sapply(clust17.cells, function(x) paste0(gsub("CMP", "CMPm2", x), "-1"))
clust10.cells <- read.table(file = "../Seuratv1_clusterCellIDs/cluster10cellIDs.txt", col.names = "clust10")
clust10.cells <- sapply(clust10.cells, function(x) paste0(gsub("CMP", "CMPm2", x), "-1"))
clust11.cells <- read.table(file = "../Seuratv1_clusterCellIDs/cluster11cellIDs.txt", col.names = "clust11")
clust11.cells <- sapply(clust11.cells, function(x) paste0(gsub("CMP", "CMPm2", x), "-1"))
```

Add new metadata column
```{r}
cmp.object@meta.data['clust.ID'] <- 0
head(cmp.object@meta.data)
```

now map new ids
```{r}
cmp.object@meta.data$clust.ID[rownames(cmp.object@meta.data) %in% clust3.cells] <- 3
cmp.object@meta.data$clust.ID[rownames(cmp.object@meta.data) %in% clust17.cells] <- 17
cmp.object@meta.data$clust.ID[rownames(cmp.object@meta.data) %in% clust10.cells] <- 10
cmp.object@meta.data$clust.ID[rownames(cmp.object@meta.data) %in% clust11.cells] <- 11
```

do numbers make sense?
```{r}
nrow(cmp.object@meta.data[cmp.object@meta.data$clust.ID == 10,])
nrow(cmp.object@meta.data[cmp.object@meta.data$clust.ID == 11,])
nrow(cmp.object@meta.data[cmp.object@meta.data$clust.ID == 17,])
nrow(cmp.object@meta.data[cmp.object@meta.data$clust.ID == 3,])
```

## Identify variable genes for new biomark
```{r}
length(cmp.object@assays$RNA@var.features)
```
```{r}
top100.var <- cmp.object@assays$RNA@var.features[1:100]
length(top100.var)
```






# Total var 90%
## Neighborhood and umap
set total.var <- 90%
```{r}
tot.var <- percent.variance(cmp.object@reductions$pca@stdev, plot.var = FALSE, return.val = TRUE)
ndims <- length(which(cumsum(tot.var) <= 90))

cmp.object <- FindNeighbors(cmp.object, dims = 1:ndims)
cmp.object <- FindClusters(cmp.object, resolution = 0.5)
cmp.object <- RunUMAP(cmp.object, dims = 1: ndims)

```
Plot UMAP

```{r}
for(x in c(0.5, 1, 1.5, 2, 2.5)){
	cmp.object <- FindClusters(cmp.object, resolution = x)
}
saveRDS(cmp.object, file = paste0(projectName, "_dim", ndims, ".RDS"))
```

```{r}
for (meta.col in colnames(cmp.object@meta.data)){
	if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
		myplot <- DimPlot(cmp.object, 
											group.by = meta.col,
											reduction = "umap", 
											cols = color.palette
											) + 
			ggtitle(paste0(projectName, " dim", ndims, "res", gsub("RNA_snn_res", "", meta.col) ))
		plot(myplot)
	}
}
```

### Clustree
what's the max resolution we can achieve while keepign clusters stable?
```{r fig.width = 10, fig.height = 5}
plot.title <- paste0(projectName, "_clustree_ndim", max(cmp.object@commands$RunUMAP.RNA.pca$dims))
my.clustree <- clustree(cmp.object, prefix = "RNA_snn_res.", node_colour = "sc3_stability", exprs = "scale.data") + 
	scale_color_continuous(low = 'red3', high = 'white') + 
	ggtitle(plot.title)
png(filename = paste0(plot.title, ".png"), height = 800, width = 1600)
plot(my.clustree)
dev.off()

```
I think I'm liking res.1.0 from this. Although how much does this change if I use fewer PCs...



for each resolution, number/percentage of cells in each cluster?

```{r}
tot.cells <- nrow(cmp.object@meta.data)
for (meta.col in colnames(cmp.object@meta.data)){
	if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
		new.clusters <- sort(as.numeric(levels(cmp.object@meta.data[[meta.col]])))
		stats.df <- data.frame(matrix(ncol = 2, nrow = length(new.clusters)))
		colnames(stats.df) <- c("num_cells", "pct_pop")
		rownames(stats.df) <- new.clusters
		meta.df <- cmp.object@meta.data
		for(row.id in rownames(stats.df)){
				num.x <- nrow(meta.df[meta.df[meta.col] == row.id,])
				pct.x <- as.integer(num.x / tot.cells *100)
				# print(pct.x)
				stats.df[row.id, "num_cells"] <- num.x
				stats.df[row.id, "pct_pop"] <- pct.x
		}
		print(stats.df)
	}
}
```



For each resolution, what percentage of cells in each cluster are enriched for one of our clust.IDs?


Test: what percentage of each new clusterID matches one of the older clusters?
```{r}
for (meta.col in colnames(cmp.object@meta.data)){
	if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
		new.clusters <- sort(as.numeric(levels(cmp.object@meta.data[[meta.col]])))
		enrich.df <- data.frame(matrix(ncol = 4, nrow = length(new.clusters)))
		colnames(enrich.df) <- c(3, 17, 10, 11)
		rownames(enrich.df) <- new.clusters
		meta.df <- cmp.object@meta.data
		for(row.id in rownames(enrich.df)){
			tot.clus <- nrow(meta.df[meta.df[[meta.col]] == row.id,])
			for(col.id in colnames(enrich.df)){
				num.x <- nrow(meta.df[(meta.df[[meta.col]] == row.id) & (meta.df$clust.ID == col.id),])
				pct.x <- as.integer(num.x / tot.clus *100)
				# print(pct.x)
				enrich.df[row.id, col.id] <- pct.x
			}
		}
		colnames(enrich.df) <- sapply(colnames(enrich.df), function(x) paste0("oldcluster", x))
		rownames(enrich.df) <- sapply(rownames(enrich.df), function(x) paste0("newcluster", x))
		xlsx::write.xlsx(enrich.df, file = paste0("PctOfNewClustersOverlappingOldClusters_", projectName, "_dim", ndims, ".xlsx"), sheetName = paste0(gsub("RNA_snn_", "", meta.col)), append = TRUE)
		print(enrich.df)
	}
}

```
Absolutely terrible overlap, no enrichment of any of these across the new clustering algorithm. Maybe should try 95% variation covered

## Find old cells on UMAP

time for the super scarey moment to see if the cells from seuratv1 still cluster together on in seurat v4

```{r fig.width = 4}
DimPlot(cmp.object,
				reduction = "umap",
				group.by = "clust.ID", 
				# split.by = "orig.ident",
				cols = c("gray", "orange", "blue", "red", "green"),)
```
```{r fig.width = 4}
DimPlot(cmp.object,
				reduction = "umap",
				group.by = "orig.ident", 
				split.by = "clust.ID",
				cols = c("gray", "orange", "blue", "red", "green"),)
```


# Total var 85%
## Neighborhood and umap
set total.var <- 85%
```{r}
tot.var <- percent.variance(cmp.object@reductions$pca@stdev, plot.var = FALSE, return.val = TRUE)
ndims <- length(which(cumsum(tot.var) <= 85))

cmp.object <- FindNeighbors(cmp.object, dims = 1:ndims)
cmp.object <- FindClusters(cmp.object, resolution = 0.5)
cmp.object <- RunUMAP(cmp.object, dims = 1: ndims)

saveRDS(cmp.object, file = paste0(projectName, "_dim", ndims, ".RDS"))
```
Plot UMAP

```{r}
for(x in c(0.5, 1, 1.5, 2, 2.5)){
	cmp.object <- FindClusters(cmp.object, resolution = x)
}
```

```{r}
for (meta.col in colnames(cmp.object@meta.data)){
	if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
		myplot <- DimPlot(cmp.object, 
											group.by = meta.col,
											reduction = "umap", 
											cols = color.palette
											) + 
			ggtitle(paste0(projectName, " dim", ndims, "res", gsub("RNA_snn_res", "", meta.col) ))
		plot(myplot)
	}
}
```

### Clustree
what's the max resolution we can achieve while keepign clusters stable?
```{r fig.width = 10, fig.height = 5}
plot.title <- paste0(projectName, "_clustree_ndim", max(cmp.object@commands$RunUMAP.RNA.pca$dims))
my.clustree <- clustree(cmp.object, prefix = "RNA_snn_res.", node_colour = "sc3_stability", exprs = "scale.data") + 
	scale_color_continuous(low = 'red3', high = 'white') + 
	ggtitle(plot.title)
png(filename = paste0(plot.title, ".png"), height = 800, width = 1600)
plot(my.clustree)
dev.off()
```



for each resolution, number/percentage of cells in each cluster?

```{r}
tot.cells <- nrow(cmp.object@meta.data)
for (meta.col in colnames(cmp.object@meta.data)){
	if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
		new.clusters <- sort(as.numeric(levels(cmp.object@meta.data[[meta.col]])))
		stats.df <- data.frame(matrix(ncol = 2, nrow = length(new.clusters)))
		colnames(stats.df) <- c("num_cells", "pct_pop")
		rownames(stats.df) <- new.clusters
		meta.df <- cmp.object@meta.data
		for(row.id in rownames(stats.df)){
				num.x <- nrow(meta.df[meta.df[meta.col] == row.id,])
				pct.x <- as.integer(num.x / tot.cells *100)
				# print(pct.x)
				stats.df[row.id, "num_cells"] <- num.x
				stats.df[row.id, "pct_pop"] <- pct.x
		}
		print(stats.df)
	}
}
```



For each resolution, what percentage of cells in each cluster are enriched for one of our clust.IDs?


Test: what percentage of each new clusterID matches one of the older clusters?
```{r}
for (meta.col in colnames(cmp.object@meta.data)){
	if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
		new.clusters <- sort(as.numeric(levels(cmp.object@meta.data[[meta.col]])))
		enrich.df <- data.frame(matrix(ncol = 4, nrow = length(new.clusters)))
		colnames(enrich.df) <- c(3, 17, 10, 11)
		rownames(enrich.df) <- new.clusters
		meta.df <- cmp.object@meta.data
		for(row.id in rownames(enrich.df)){
			tot.clus <- nrow(meta.df[meta.df[[meta.col]] == row.id,])
			for(col.id in colnames(enrich.df)){
				num.x <- nrow(meta.df[(meta.df[[meta.col]] == row.id) & (meta.df$clust.ID == col.id),])
				pct.x <- as.integer(num.x / tot.clus *100)
				# print(pct.x)
				enrich.df[row.id, col.id] <- pct.x
			}
		}
		colnames(enrich.df) <- sapply(colnames(enrich.df), function(x) paste0("oldcluster", x))
		rownames(enrich.df) <- sapply(rownames(enrich.df), function(x) paste0("newcluster", x))
		xlsx::write.xlsx(enrich.df, file = paste0("PctOfNewClustersOverlappingOldClusters_", projectName, "_dim", ndims, ".xlsx"), sheetName = paste0(gsub("RNA_snn_", "", meta.col)), append = TRUE)
		print(enrich.df)
	}
}

```
Absolutely terrible overlap, no enrichment of any of these across the new clustering algorithm. Maybe should try 95% variation covered

## Find old cells on UMAP

time for the super scarey moment to see if the cells from seuratv1 still cluster together on in seurat v4

```{r fig.width = 4}
DimPlot(cmp.object,
				reduction = "umap",
				group.by = "clust.ID", 
				# split.by = "orig.ident",
				cols = c("gray", "orange", "blue", "red", "green"),)
```
```{r fig.width = 4}
DimPlot(cmp.object,
				reduction = "umap",
				group.by = "orig.ident", 
				split.by = "clust.ID",
				cols = c("gray", "orange", "blue", "red", "green"),)
```



# Total var 95%

## Neighborhood and umap
set total.var <- 95%
```{r}
tot.var <- percent.variance(cmp.object@reductions$pca@stdev, plot.var = FALSE, return.val = TRUE)
ndims <- length(which(cumsum(tot.var) <= 95))

cmp.object <- FindNeighbors(cmp.object, dims = 1:ndims)
cmp.object <- FindClusters(cmp.object, resolution = 0.5)
cmp.object <- RunUMAP(cmp.object, dims = 1: ndims)

saveRDS(cmp.object, file = paste0(projectName, "_dim", ndims, ".RDS"))
```
Plot UMAP

```{r}
for(x in c(0.5, 1, 1.5, 2, 2.5)){
	cmp.object <- FindClusters(cmp.object, resolution = x)
}
```

```{r}
cmp.object <- readRDS("CMP_dim41.RDS")
```


```{r fig.width = 10, fig.height = 5}
plot.title <- paste0(projectName, "_clustree_ndim", max(cmp.object@commands$RunUMAP.RNA.pca$dims))
my.clustree <- clustree(cmp.object, prefix = "RNA_snn_res.", node_colour = "sc3_stability", exprs = "scale.data") + 
	scale_color_continuous(low = 'red3', high = 'white') + 
	ggtitle(plot.title)
png(filename = paste0(plot.title, ".png"), height = 800, width = 1600)
plot(my.clustree)
dev.off()
```

For each resolution, what percentage of cells in each cluster are enriched for one of our clust.IDs?


Test: what percentage of each new clusterID matches one of the older clusters?
```{r}
for (meta.col in colnames(cmp.object@meta.data)){
	if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
		new.clusters <- sort(as.numeric(levels(cmp.object@meta.data[[meta.col]])))
		enrich.df <- data.frame(matrix(ncol = 4, nrow = length(new.clusters)))
		colnames(enrich.df) <- c(3, 17, 10, 11)
		rownames(enrich.df) <- new.clusters
		meta.df <- cmp.object@meta.data
		for(row.id in rownames(enrich.df)){
			tot.clus <- nrow(meta.df[meta.df[[meta.col]] == row.id,])
			for(col.id in colnames(enrich.df)){
				num.x <- nrow(meta.df[(meta.df[[meta.col]] == row.id) & (meta.df$clust.ID == col.id),])
				pct.x <- as.integer(num.x / tot.clus *100)
				# print(pct.x)
				enrich.df[row.id, col.id] <- pct.x
			}
		}
		colnames(enrich.df) <- sapply(colnames(enrich.df), function(x) paste0("oldcluster", x))
		rownames(enrich.df) <- sapply(rownames(enrich.df), function(x) paste0("newcluster", x))
		xlsx::write.xlsx(enrich.df, file = paste0("PctOfNewClustersOverlappingOldClusters_", projectName, "_dim", ndims, ".xlsx"), sheetName = paste0(gsub("RNA_snn_", "", meta.col)), append = TRUE)
		print(enrich.df)
	}
}

```
Absolutely terrible overlap, no enrichment of any of these across the new clustering algorithm. Maybe should try 95% variation covered

## Find old cells on UMAP

time for the super scarey moment to see if the cells from seuratv1 still cluster together on in seurat v4

```{r fig.width = 2}
DimPlot(cmp.object,
				reduction = "umap",
				group.by = "clust.ID", 
				pt.size = .1,
				# split.by = "orig.ident",
				cols = c("gray", "orange", "blue", "red", "green"),)
```
```{r fig.width = 4}
DimPlot(cmp.object,
				reduction = "umap",
				group.by = "orig.ident", 
				split.by = "clust.ID",
				cols = c("gray", "orange", "blue", "red", "green"),)
```



### Gene expression of old clustrs on new map
Let's see if we can get some gene expression profiles on these...
```{r, fig.height=10, fig.width=18}
gene.list <- c("Gata1", "Gata2", "Pf4", "Dntt", "Mpo", "Meis1", "Irf8", "Elane", "Fli1", "Zfpm1")
VlnPlot(cmp.object, features = gene.list, group.by = "clust.ID", pt.size = 0.01, cols = c("gray", "orange", "blue", "red", "green"))
```


Used the exce doc to do some fancy conditional formatting. Old cluster 17 is pretty dispersed until you it resolution 2.5. Otherise, cells in old cluster 17 do not constitute more than 40% of any cells in the new clusters.  
As far as I can see, the two approaches are to do DGEof new CMP w/ resolution = 2.5, AND/OR do DGe using older cluster IDs. Sure seems to make sense to do both...


# DGE w/ resolution = 2.5
Strt with comparing all clusters against all other clusters
Write out cluster info


calculate `FindAllMarkers()` for different idents and save to new file
```{r}
ident.list <- c("RNA_snn_res.0.5", "RNA_snn_res.1", "RNA_snn_res.1.5", "RNA_snn_res.2", "RNA_snn_res.2.5", "clust.ID")
for(tested.ident in ident.list){
	Idents(cmp.object) <- tested.ident
	all.markers <- FindAllMarkers(cmp.object)
	xlsx::write.xlsx(x = all.markers[,c("avg_log2FC", "p_val_adj", "cluster", "gene")], 
									 file = paste0(projectName, "_FindALLMarkers_res2.5.xlsx"), 
									 sheetName = tested.ident, 
									 col.names = TRUE, 
									 row.names = FALSE, 
									 append = TRUE)
}
```

Create `FindAllMarkers()` lists for GSEA
```{r }
Idents(cmp.object) <- "RNA_snn_res.2.5"
res.2.5.allmarkers <- FindAllMarkers(cmp.object)
```

## Map HGNC symbols
```{r}
Mouse2HumanTable <- Mouse2Human(res.2.5.allmarkers$gene)

HGNC <- with(Mouse2HumanTable, Mouse2HumanTable$HGNC[match(res.2.5.allmarkers$gene, Mouse2HumanTable$MGI)])
head(res.2.5.allmarkers)
res.2.5.allmarkers$HGNC <- HGNC
tail(res.2.5.allmarkers)
sig.res.2.5 <- res.2.5.allmarkers[res.2.5.allmarkers$p_val_adj <= 0.05, ]
sig.res.2.5 <- sig.res.2.5[c("avg_log2FC", "HGNC", "cluster")]
sig.res.2.5 <- sig.res.2.5[!(sig.res.2.5$HGNC == "" | is.na(sig.res.2.5$HGNC)),] # GSEA will fail if there are any blanks or NAs in the table
sig.res.2.5 <- sig.res.2.5[]

```


```{r}
for(cluster in unique(sig.res.2.5$cluster)){
	print(paste("writing cluster", cluster))
	new.table <- sig.res.2.5[sig.res.2.5$cluster == cluster, c("HGNC", "avg_log2FC")]
	new.table <- new.table[order(-new.table$avg_log2FC), ]
	write.table(new.table, file = paste0("RankList_res2.5_findAll_hgnc/res.2.5cluster", cluster, ".rnk"), quote = FALSE, row.names = FALSE, col.names = TRUE, sep = "\t", )
	
}
```



calculate `FindMarkers()` that distinguish each cluster (might overlab between clusters)
```{r}
ident.list <- c("RNA_snn_res.0.5", "RNA_snn_res.1", "RNA_snn_res.1.5", "RNA_snn_res.2", "RNA_snn_res.2.5", "clust.ID")
for(tested.ident in ident.list){
	object.copy <- cmp.object
	Idents(object.copy) <- tested.ident
	print(paste("testing", tested.ident))
	for (cluster in sort(as.numeric(levels(object.copy@meta.data[[tested.ident]])))){
		print(paste("looking at cluster", cluster))
		cluster.markers <- FindMarkers(object.copy, ident.1 = cluster)
		try(
			xlsx::write.xlsx(x = cluster.markers[,c("avg_log2FC", "p_val_adj")], 
											 file = paste0(projectName, "_FindMarkers_", gsub("RNA_snn_", "", tested.ident), ".xlsx"), 
											 sheetName = paste0("clst", cluster), 
											 col.names = TRUE, 
											 row.names = TRUE, 
											 append = TRUE)
		)	
	}
	remove(object.copy)
}
```



```{r}
for (cluster in sort(as.numeric(levels(cmp.object@meta.data$RNA_snn_res.2.5)))){
	cluster.markers <- FindMarkers(cmp.object, ident.1 = cluster)
	xlsx::write.xlsx(x = cluster.markers[,c("avg_log2FC", "p_val_adj")], 
									 file = paste0(projectName, "_FindMarkers_res2.5.xlsx"), 
									 sheetName = paste0("clst", cluster), 
									 col.names = TRUE, 
									 row.names = TRUE, 
									 append = TRUE)
}
```

## Combine clusters that might represent old cluster ids

# DGE w/ metadata against clust.ID against "0"
reset ident as "clust.ID" and rerun `FindAllMarkers()`
```{r}
	Idents(cmp.object) <- "clust.ID"
	all.markers <- FindAllMarkers(cmp.object)
	xlsx::write.xlsx(x = all.markers[,c("avg_log2FC", "p_val_adj", "cluster", "gene")], 
									 file = paste0(projectName, "_FindALLMarkers_clustID.xlsx"), 
									 sheetName = "clustID", 
									 col.names = TRUE, 
									 row.names = FALSE, 
									 append = TRUE)
```


```{r}
# Idents(cmp.object) <- "clust.ID"
for (cluster in unique(cmp.object@meta.data$clust.ID)){
	print(cluster)
	cluster.markers <- FindMarkers(cmp.object, ident.1 = cluster)
	xlsx::write.xlsx(x = cluster.markers[,c("avg_log2FC", "p_val_adj")], 
									 file = paste0(projectName, "_FindMarkers_clustID.xlsx"), 
									 sheetName = paste0("oldclust", cluster), 
									 col.names = TRUE, 
									 row.names = TRUE, 
									 append = TRUE)
}

```


# Distinguishing features of clusters
Previously defined biomark genes based on PC contributions. Original list was based on *all* msAggr, but let's see how CMP subset does?
```{r fig.height = 30, fig.width=6}
VizDimLoadings(cmp.object, dims = 1:10, nfeatures = 30, reduction = "pca", ncol = 2)
```

```{r}
pca.df <- cmp.object[["pca"]]
pca.df <- as.data.frame(as.matrix(slot(object = pca.df, name = "feature.loadings")))
print(cmp.object[["pca"]], dims = 2, nfeatures = 5)
rownames(pca.df[pca.df$PC_2 %in% sort(pca.df$PC_2, decreasing = TRUE)[1:5], ])
rownames(pca.df[pca.df$PC_2 %in% sort(pca.df$PC_2)[1:5], ])
```

now we can get a list of principal components!  
first pull the list of oldAnalysis CMP top PC genes
```{r}
cmp.biomark <- read.table(file = "/Users/heustonef/Desktop/CMPSubpops/BioMark/ProbePanels/CMP_PCTopGenes.txt", sep = "\t", header = TRUE)
biomark.cmptargets <- c()
for(df.col in 1:ncol(cmp.biomark)){
	biomark.cmptargets <- c(biomark.cmptargets, biomark[,df.col])
}
print(colnames(biomark))
print(paste("total gene count:", length(biomark.cmptargets)))
```

Now get the list of current pc gene trgets (oldAnalysis used ndim = 1:6, so we'll start with that range)
```{r}
pc.list <- c("PC_1", "PC_2", "PC_3", "PC_4", "PC_5", "PC_6")
pc.genes <- lapply(pc.list, function(x) rownames(pca.df[pca.df[[x]] %in% sort(pca.df[[x]], decreasing = TRUE)[1:30],])) #targeting roughly 180 genes like in biomark.cmptargets
pc.genes <- unique(unlist(pc.genes))
print(paste("total gene count:", length(pc.genes)))
```

Now compare the lists, I guess:

```{r}
# setdiff(x,y) gives you things in x not in y. setdiff(y,x) gives you things in y not in x
setdiff(biomark.cmptargets, pc.genes)
# print(paste("\n length:", length(setdiff(biomark.cmptargets, pc.genes))))
writeLines(c("", "length:", length(setdiff(biomark.cmptargets, pc.genes))))
```
Umm, yeah that went kinda how I expected. Let's do this again, but for the actual biomark gene lists.
```{r}
biomark <- read.table(file = "/Users/heustonef/Desktop/CMPSubpops/BioMark/ProbePanels/BiomarkProbeList.txt", sep = "\t")
biomark <- biomark[,1]
setdiff(biomark, pc.genes)
writeLines(c("", "length:", length(setdiff(biomark, pc.genes))))
```


What if we increase the number of pcs but decrease the depth of each? This might cover more of `biomark	`, which was originally developed using msAggr instead of only the CMP subset
```{r}
pc.list <- c("PC_1", "PC_2", "PC_3", "PC_4", "PC_5", "PC_6", "PC_7", "PC_8", "PC_9", "PC_10")
pc.genes <- lapply(pc.list, function(x) rownames(pca.df[pca.df[[x]] %in% sort(pca.df[[x]], decreasing = TRUE)[1:20],]))
pc.genes <- unique(unlist(pc.genes))
print(paste("total gene count:", length(pc.genes)))

```
```{r}
setdiff(biomark, pc.genes)
writeLines(c("", "length:", length(setdiff(biomark, pc.genes))))
```

For comparison, let's just see how many of `biomark.cmptargets` were actually included in `biomark`
```{r}
setdiff(biomark.cmptargets, biomark)
writeLines(c("", "length:", length(setdiff(biomark.cmptargets, pc.genes))))
```
```{r}
length(biomark) - length(setdiff(biomark, biomark.cmptargets))
```
```{r}
length(biomark) - length(setdiff(biomark, pc.genes))
```
So when you look at it like that, it's not actually that far off.


What are the similarities?:
```{r}
setdiff(setdiff(biomark, biomark.cmptargets), setdiff(biomark, pc.genes))
```
These are genes from the 97probes not in the old CMP set that are also not in the new CMP set. Other than Itga2b (which is a failed probe anyway), nothing screams. Also we'd have thrown Flt3 and Cd34 for in anyway because they're requisite cell surface markers (also Flt3 surface marker is expensive but otherwise not noteworthy and not used in the current sorting strategy)

What about cell surface marker expression?
* Cd34
* Cd16/32
* Cd9
* Cd41
* Cd48
* Sca1 (just throw that in for sh*&s and giggles)
```{r, fig.height = 15, fig.width=10}
surface.markers <- c("Cd34", "Fcgr3", "Fcgr2b", "Cd9", "Itga2b", "Cd48", "Ly6a")
FeaturePlot(cmp.object, features = surface.markers, pt.size = 1, split.by = "clust.ID", ncol = 1)
```
Save as png
```{r}
png(filename = "FeaturePlot_CMP_surfaceMarkers_clustIDfacet.png", height = 1600, width = 1600)
FeaturePlot(cmp.object, features = surface.markers, pt.size = 1, split.by = "clust.ID", ncol = 1)
dev.off()
```







# Compare @g hierarchcial clusteirng

Do clustering using biomark RNAs as input
```{r}
# Read in BiomarkRNAs
biomark.rnas <- read.table('/Users/heustonef/Desktop/10XGenomicsData/BiomarkRNAs.txt')
biomark.rnas <- biomark.rnas$V1
```

use biomark RNAs to define dimensional reduction
```{r}
cmp.object <- readRDS("CMP_raw.RDS")
cmp.object <- RunPCA(cmp.object, features = biomark.rnas, ndims.print = 1:5, , nfeatures.print = 5)
ElbowPlot(cmp.object, ndims = 50)
```


Now run the clustering
```{r}
tot.var <- percent.variance(cmp.object@reductions$pca@stdev, plot.var = FALSE, return.val = TRUE)
ndims <- length(which(cumsum(tot.var) <= 90))

cmp.object <- FindNeighbors(cmp.object, dims = 1:ndims)
cmp.object <- FindClusters(cmp.object, resolution = 0.5)
cmp.object <- RunUMAP(cmp.object, dims = 1: ndims)


```

find the clusters

```{r}
for(x in c(0.5, 1, 1.5, 2, 2.5)){
	cmp.object <- FindClusters(cmp.object, resolution = x)
}
```

Plot the umaps and cell cluster ids
```{r}
for (meta.col in colnames(cmp.object@meta.data)){
	if(grepl(pattern = ("RNA_snn_res"), x = meta.col)==TRUE){
		myplot <- DimPlot(cmp.object, 
											group.by = meta.col,
											reduction = "umap", 
											cols = color.palette
											) + 
			ggtitle(paste0(projectName, " dim", ndims, "res", gsub("RNA_snn_res", "", meta.col) ))
		plot(myplot)
	}
}
```
### Calculate anticipated number of cells you'll find in each biomark cluster
Get # cells in each cluster

```{r}
tot.cellcount <- nrow(cmp.object@meta.data)
res05.list <- sort(unique(cmp.object@meta.data$RNA_snn_res.0.5), decreasing = FALSE)
sapply(res05.list, 
			 function(x){
			 	print(
			 		paste(
			 			"cluster", x, "=", 
			 			nrow(cmp.object@meta.data[cmp.object@meta.data$RNA_snn_res.0.5 == x,]), 
			 			"cells or", 
			 			round(nrow(cmp.object@meta.data[cmp.object@meta.data$RNA_snn_res.0.5 == x,])/tot.cellcount*100, digits = 2), 
			 			"% of total"
			 		)
			 	)
			 }
			)
```

So we did the dimensional reduction based on the biomark RNAs, then did our UMAP nearest neighbor clustering.


In the biomark hierarchcial clustering analysis I assayed 167 cells. The smallest cluster I detected had 3 cells, or 1.8% of total, and this is an uncomfortably small number of cells. Based on the UMAP calculations I would therefore expect to find 11 or 12 of the predicted 15 clusters. I found 12, and I don't really like that last one, so 11 or 12. Since I did the hierarchcial clustering yesterday and did this math today, we can say it was independent of these results and therefore totally legit. Yay!!

